MIR-21 promoter driven targeted cancer therapy

ABSTRACT

The invention provides a nucleic acid construct comprising a promoter sequence derived from microRNA-21 (miR-21) linked to a nucleic acid sequence encoding an anti-cancer agent, an example of which is a toxin. The constructs of the invention are particularly useful for treating tumors expressing miR-21.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/145,332, filed on Nov. 17, 2011, which is a national phase filingunder 35 U.S.C. 371 of International Application No. PCT/IL2010/000049,filed on Jan. 19, 2010, which claims the benefit of U.S. ProvisionalApplication No. 61/145,751, filed on Jan. 20, 2009, the entirety ofthese applications is hereby incorporated herein by reference for theteachings therein.

FIELD OF THE INVENTION

The present invention is directed to the field of cancer treatment, andprovides a nucleic acid construct particularly useful for treatingtumors expressing miR-21.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are an abundant class of short (20-24 nt) endogenousnoncoding RNAs that act as post-transcriptional regulators of geneexpression by base-pairing with their target mRNAs (for review see forexample, Bartel, Cell 2004, 116:281-297). In general, genes encodingmiRNAs are transcribed by RNA polymerase II as part of capped andpolyadenylated primary transcripts (pri-miRNAs) that can be eitherprotein-coding or non-coding. The primary transcript is cleaved by theDrosha ribonuclease III enzyme to produce an approximately 70 ntstem-loop precursor miRNA (pre-miRNA), which is further cleaved by thecytoplasmic Dicer ribonuclease to generate the mature miRNA andantisense miRNA star (miRNA*) products. The mature miRNA is incorporatedinto a RNA-induced silencing complex (RISC), which recognizes targetmRNAs through imperfect base pairing with the miRNA and most commonlyresults in translational inhibition or destabilization of the targetmRNA.

To date, several hundred miRNAs have been described in humans and alarge number of them have been implicated in cancer. miRNAs that havebeen experimentally shown directly to induce tumor phenotypes (i.e.,formation) have been termed “oncomirs”. MiR-21 (also known as MIRN21,miRNA21, hsa-mir-21 and MIR21) is such an oncomir shown to targetmultiple tumor/metastasis suppressor genes and to have a role in tumorgrowth, invasion and metastasis. Various studies show that miR-21 isexclusively expressed in cancerous cell lines and solid human tumors,but not in non-transformed cell lines or in the adjacent non canceroustissue (see for example, Iorio et al., Cancer Res, 2005. 65(16):7065-7070; Si et al., Oncogene, 2007. 26(19): 2799-2803; Volinia et al.,Proc Natl Acad Sci USA, 2006. 103(7): 2257-2261). Thus, evaluation ofmiR-21 expression has been suggested in cancer diagnosis (see, forexample, U.S. Patent Application Publication No. 2006/0105360).

U.S. Patent Application Publication No. 2008/0306018 discloses methodsfor diagnosing pancreatic cancer comprising measuring the level of atleast one miRNA gene product, including miR-21, in a test sample fromthe subject. This publication further discloses a method of treatingpancreatic cancer in a subject in which at least one miRNA gene productis downregulated or upregulated in the cancer cells relative to controlcells, inter alia when miR-21 is upregulated in the cancer cells, themethod comprising administering to the subject an effective amount of atleast one compound for inhibiting expression of the at least one miRNAgene product, such that proliferation of cancer cells in the subject isinhibited. Also disclosed are pharmaceutical compositions for treatingpancreatic cancer, comprising at least one isolated miRNA gene productor an inhibitor thereof.

Similar methods and compositions have been disclosed for the treatmentof lung cancer (U.S. Patent Application Publication No. 2008/0306017),breast cancer (U.S. Patent Application Publication No. 2008/0261908) andsolid cancers such as prostate cancer, stomach cancer, pancreaticcancer, lung cancer, breast cancer and colon cancer (U.S. PatentApplication Publication No. 2008/0306006). Other exemplary publicationswhich suggest elevating or inhibiting the expression of miR-21 includeU.S. Patent Application Publication Nos. 2008/0171715, 2008/0199961 and2008/0050744. Certain attempts to use miRNAs to control tissue tropismhave also been reported (see for example, Barnes et al., Cell HostMicrobe, 2008. 4(3):239-248).

Selective targeting of a specific cancer using differentially expressedgenes has been previously demonstrated successfully where regulatorysequences of the H19 gene differentially expressed in bladder cancerwere used to control the expression of diphtheria toxin (Ohana et al., JGene Med, 2005. 7(3): 366-374).

WO 99/18195 and U.S. Pat. No. 7,041,654 teach the specific expression ofheterologous sequences, particularly genes encoding cytotoxic products(e.g. diphtheria toxin), in tumor cells under the control of a cancerspecific promoter (e.g., H19 and IGF promoters).

WO 2007/034487 discloses a nucleic acid construct comprising: (i) afirst nucleic acid sequence encoding TNF alpha; (ii) a second nucleicacid sequence encoding a Diphtheria toxin; and (iii) at least oneadditional nucleic acid sequence comprising a cancer specific promoter(e.g. H19 and IGF promoters); the TNF alpha and Diphtheria toxinencoding sequences being under an expression control of the cancerspecific promoter. Also disclosed are construct systems and methods anduses thereof.

WO 2008/087642 discloses compositions and methods for the treatment ofcancer and other conditions that are associated with elevated expressionof the H19 gene, utilizing constructs encoding H19-silencing nucleicacid agents such as inhibitory RNA.

WO 2006/065938 discloses, inter alia, a method for treating cancer in amammal comprising administering to the mammal an effective amount of anucleic acid that encodes an anticancer agent operably linked to aSPANX-N1 promoter.

WO 2003/093441 discloses a method of inhibiting expression of a gene ina cell comprising introducing into said cell a DNA construct comprisinga promoter functional in said cell operably linked to a nucleic acidsequence encoding an miRNA precursor having a stem loop structure andcomprising in said stem a sequence complementary to a portion of an RNAtranscript of said gene, wherein, following introduction of saidconstruct into said cell, said nucleic acid sequence is transcribed andprocessed so that said miRNA precursor is produced. Further disclosed isa plasmid construct comprising precursor miRNA-21 linked to a CMVpromoter.

Cai et al. disclose a plasmid containing the firefly luciferase genelinked to an ˜1 kb DNA fragment of the pri-miR-21 transcription unitcorresponding to the region from −959 to +49 relative to the T1transcription start site (Cai et al., RNA (2004) 10:1957-1966).According to this disclosure, firefly luciferase activity was detectedin 293T cells transfected with such a plasmid, leading the authors toconclude that sequences located 5′ to the pri-miR-21 transcription unitcan function as an mRNA promoter.

Fujita and Iba disclose a putative promoter region of miR-21 462nucleotides in length (Fujita and Iba Bioinformatics 2008,24(3):303-308), and Fujita et al disclose the miR-21 promoter includingbinding sites for activation protein 1 and PU.1 (Fujita et al., J MolBiol 2008 May 2; 378(3):492-504).

While several therapeutic approaches utilizing gene therapy in cancerpatients have been suggested, there exists a need for additionalefficacious anti-cancer agents and vectors. As tumors are known toexhibit significant genomic instability and heterogeneity, the use ofhitherto known gene therapy vectors is likely to fail in a substantialnumber of the patients.

Nowhere in the prior art is it taught or suggested that a miR-21promoter can be used in recombinant nucleic acid constructs forexpressing selectively cytotoxic agents in cancer cells. Nor does theart demonstrate the use of such vectors as an effective and safeanti-cancer treatment in vivo. There remains an unmet medical need fordeveloping gene therapy vectors having enhanced therapeutic activity,minimized toxicity and a broad target range for treating neoplasticdisorders.

SUMMARY OF THE INVENTION

The invention is directed to nucleic acid constructs and methods forproviding targeted cancer therapy. Specifically, the constructs of theinvention comprise at least one nucleic acid sequence encoding acytotoxic or cytostatic anti-cancer molecule, such as a toxin, thenucleic acid sequence being operably linked to an miR-21transcription-regulating sequence. Vectors comprising these constructs,pharmaceutical compositions comprising them and use thereof for treatingcancers and solid tumors are also provided.

The present invention discloses for the first time novel nucleic acidconstructs and vectors in which expression of an anti-cancer agent isplaced under the transcriptional control of an miR-21 promoter sequence.The inventors of the present invention have surprisingly demonstratedthat such constructs have activity in depressing de novo proteinsynthesis in cancer cells in vitro, and moreover inhibit tumor growthand metastasis in vivo.

The invention is based, in part, on the finding that direct injectioninto tumors induced in mice, of a nucleic acid construct having anmiR-21 promoter operatively linked to a nucleic acid sequence encodingdiphtheria toxin, results either in complete eradication of the tumor orsubstantial inhibition of tumor growth.

Thus, in a first aspect the present invention provides a nucleic acidconstruct comprising a miR-21 promoter sequence and at least one nucleicacid sequence encoding an anti-cancer agent, wherein the nucleic acidsequence encoding the anti-cancer agent is operably linked to the miR-21promoter sequence.

In particular embodiments, the nucleic acid construct is the product ofrecombinant methods, a chemical synthesis or a combination thereof.

In particular embodiments, the miR-21 promoter sequence has at least 80%identity with SEQ ID NO: 1. In particular embodiments, the miR-21promoter sequence has at least 85%, or at least 90% identity, or atleast 95% identity with SEQ ID NO: 1. In particular embodiments, themiR-21 promoter sequence comprises SEQ ID NO: 1.

In particular embodiments, the anti-cancer agent is selected fromcytotoxic and cytostatic proteins and peptides capable of killing atarget cell or inhibiting its growth or proliferation. In particularembodiments, the anti-cancer agent is selected from a toxin, adrug-metabolizing enzyme which converts a prodrug to a drug havingcytotoxic activity, an inducer of apoptosis, and a combination thereof.In particular embodiments, the toxin is selected from the groupconsisting of a bacterial toxin, a plant toxin, a fungal toxin, and acombination thereof. In particular embodiments, the bacterial toxin isselected from the group consisting of diphtheria toxin, Pseudomonasexotoxin, cholera toxin, anthrax toxin, botulinum toxin, pertussistoxin, E. coli enterotoxin, and shiga toxin. In particular embodiments,the plant toxin is selected from the group consisting of ricin,modeccin, abrin, volkensin and viscumin. In particular embodiments, thefungal toxin is selected from the group consisting of α-sarcin,restrictocin, mitogillin, enomycin, RNase T1 and phenomycin.

In particular embodiments, the toxin comprises a cytotoxic fragment of anaturally occurring toxin. In particular embodiments, the cytotoxicfragment of a toxin is fragment A of diphtheria toxin (DT-A). Inparticular embodiments, DT-A has the amino acid sequence of SEQ ID NO:19. In particular embodiments, the nucleic acid sequence encoding DT-Acomprises SEQ ID NO: 18.

In particular embodiments, the drug-metabolizing enzyme is a kinase. Inparticular embodiments, the kinase is a thymidine kinase. In particularembodiments, the prodrug target of the thymidine kinase is ganciclovir.In particular embodiments, the kinase is a viral thymidine kinase. Inparticular embodiments, the inducer of apoptosis is selected from thegroup consisting of PUMA; BAX; BAK; BcI-XS; BAD; BIM; BIK; BID; HRK; AdE1B; an ICE-CED3 protease; TRAIL; SARP-2; and apoptin.

In a particular embodiment, the construct comprises SEQ ID NO: 1 as themiR-21 promoter sequence and further comprises SEQ ID NO: 18 as thenucleic acid sequence encoding an anti-cancer agent. In particularembodiments, the nucleic acid sequence comprising an miR-21 promotersequence is substantially devoid of a nucleic acid sequencecorresponding to or complementary to an miR-21 transcription orprocessing product. In particular embodiments, the nucleic acidconstruct is substantially devoid of a nucleic acid sequencecorresponding to or complementary to a form of miR-21 selected from thegroup consisting of: a primary transcript of miR-21 (pri-miR-21); aprecursor of miR-21 (pre-miR-21); an RNA duplex of miR-21, and a maturemiR-21. In particular embodiments, the nucleic acid construct issubstantially devoid of a nucleic acid sequence corresponding to orcomplementary to a nucleic acid sequence selected from the groupconsisting of: SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5and SEQ ID NO: 26.

In a particular embodiment, the nucleic acid construct comprises asequence selected from the group consisting of SEQ ID NO: 7 and SEQ IDNO: 8.

In a particular embodiment, the miR-21 promoter sequence is a variant ofa native miR-21 promoter, such as a homolog, analog or fragment of anative miR-21 promoter.

In another aspect, the invention provides a vector comprising therecombinant nucleic acid construct of the invention.

In another aspect, the invention provides an isolated host cellcomprising the vector of the invention.

In a particular embodiment, the vector is selected from the groupconsisting of SEQ ID NO: 9 and SEQ ID NO: 10.

In a particular embodiment, the construct encodes a fusion proteincomprising the anti-cancer agent. In a particular embodiment, theconstruct further comprises a nucleic acid sequence that encodes anantibody or a fragment thereof comprising at least the antigen bindingportion of the antibody, wherein the antibody or antibody fragment isspecific for a cancer-related protein. In a particular embodiment, theconstruct comprises a nucleic acid sequence encoding a fusion proteinwherein the fusion protein comprises segments corresponding to theanti-cancer agent and to the antibody or antibody fragment.

In another aspect, the invention provides a pharmaceutical compositioncomprising as an active ingredient at least one nucleic acid constructof the invention and a pharmaceutically acceptable carrier, excipient ordiluent.

In another embodiment, the present invention provides a method fortreating cancer in a human subject, the method comprising administeringto a human subject in need thereof a therapeutically effective amount ofa nucleic acid construct of the present invention, thereby treatingcancer in the human subject.

In another embodiment, the present invention provides a method forinhibiting tumor progression in a human subject, the method comprisingadministering to a human subject in need thereof a therapeuticallyeffective amount of a nucleic acid construct of the present invention,thereby inhibiting tumor progression in the human subject.

In another embodiment, the present invention provides a method forinhibiting tumor metastasis in a human subject, the method comprisingadministering to a human subject in need thereof a therapeuticallyeffective amount of a nucleic acid construct of the present invention,thereby inhibiting tumor metastasis in the human subject.

In another embodiment, the present invention provides a method forreducing or alleviating a symptom associated with a neoplastic disorderin a human subject, the method comprising administering to a humansubject in need thereof a therapeutically effective amount of a nucleicacid construct of the present invention, thereby reducing or alleviatinga symptom associated with a neoplastic disorder in the human subject.

In the methods of the invention, said subject is afflicted, in oneembodiment, with a cancer, tumor or a neoplastic disorder characterizedby endogenous expression of miR-21 in at least a portion of the cellsthereof.

In particular embodiments, the administering is carried out by a routeselected from the group consisting of injection, infusion and directinjection into the tumor.

In particular embodiments, the administering comprises administering asingle dose or multiple doses of the nucleic acid construct.

In particular embodiments, the methods further comprise a step ofdetermining the level of miR-21 transcriptional activity in a biologicalsample e.g. cells or tissue, from the subject.

In particular embodiments, the methods are carried out in conjunctionwith at least one additional cancer therapy modality. In particularembodiments, the additional cancer therapy modality is selected from thegroup consisting of bone marrow transplant, cord blood cell transplant,surgery, chemotherapy, radiation therapy, immunotherapy and acombination thereof.

In another aspect, the present invention provides use of a nucleic acidconstruct of the invention for the preparation of a medicament fortreating cancer in a human subject.

In another aspect, the present invention provides use of a nucleic acidconstruct of the invention for the preparation of a medicament forinhibiting tumor progression in a human subject.

In another aspect, the present invention provides use of a nucleic acidconstruct of the invention for the preparation of a medicament forinhibiting tumor metastasis in a human subject.

In another aspect, the present invention provides use of a nucleic acidconstruct of the invention for the preparation of a medicament forreducing or alleviating a symptom associated with a neoplastic disorder.

In another aspect, the present invention provides a kit containing i)one or more dosage units of a nucleic acid construct of the inventionsufficient for one or more courses of treatment for a cancer, tumor orneoplasm expressing miR-21; and ii) instructions for administering saidnucleic acid construct to a subject in need thereof.

The compositions, methods and kits of the present invention are usefulin the treatment of a variety of cancers and neoplastic disordersassociated with expression of miR-21. In a particular embodiment, thecancer is selected from the group consisting of a sarcoma, a carcinoma,an adenocarcinoma, a lymphoma, and a leukemia. In a particularembodiment, the cancer is selected from the group consisting of breastcancer, colon cancer, hepatocellular carcinoma, cervical cancer,cholangiocarcinoma, endometrioid ovarian carcinoma, esophageal cancer,glioblastoma, head and neck cancer, leukemia, lymphoma, lung cancer,multiple myeloma, pancreatic cancer, osteosarcoma, pituitary tumor,prostate cancer, stomach cancer, and uterine leiomyoma. In a particularembodiment, the cancer is selected from the group consisting of breastcancer, colon cancer and hepatocellular carcinoma.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of the nucleic acid constructof the invention, in which a nucleic acid sequence encoding ananti-cancer agent (“killer gene”) is operably linked to a miR-21promoter sequence.

FIG. 2 demonstrates miR-21 promoter-driven expression of a reporterluciferase gene in different cell lines. A vector containing the fireflyluciferase gene (Luc) under the control of the miR-21 promoter(miR-21-Luc; SEQ ID NO: 11; black bars), and a control vector containingthe firefly luciferase gene under the control of the TATA box sequenceof the miR-21 promoter (TATA-Luc; SEQ ID NO: 20; white bars) were eachtransfected into different cell lines. The cell lines tested includedChinese hamster ovary (CHO, right bars) and the human colorectal cancercell lines HCT116 (left bars) and COLO320 (middle bars). Y axis—relativeluciferase activity.

FIG. 3 demonstrates inhibition of Renilla luciferase expression in amodel of reduction of de novo protein synthesis induced by constructsencoding a fragment of diphtheria toxin (DT-A). Cells wereco-transfected with a plasmid encoding Renilla luciferase under thecontrol of an SV-40 promoter (SEQ ID NO: 28) and either a plasmidencoding DT-A under the control of the miR-21 promoter (miR-21 Pr DT-A;SEQ ID NO: 9), or a plasmid encoding DT-A under the control of theTATA-box sequence of the miR-21 promoter (miR-21 TATA box DT-A (SEQ IDNO: 13). Cells were harvested 72 hours post-transfection and subjectedto luciferase assay.

FIG. 4 demonstrates inhibition of expression of Renilla luciferase orgreen fluorescent protein (GFP) in models of reduction of de novoprotein synthesis.

FIG. 4A (white bars) shows the results of co-transfection experiments inwhich cells were co-transfected with miR-21 Pr DT-A (SEQ ID NO: 9) ormiR-21 pr PUMA (a vector encoding the p53 up-regulated modulator ofapoptosis (PUMA) under the control of the miR-21 promoter; SEQ ID NO:10), together with a reporter plasmid encoding Renilla luciferase. FIG.4A (black bars) shows the results of co-transfection experiments incontrol systems in which cells were co-transfected with miR-21 TATA boxDT-A (SEQ ID NO: 13) or TATA PUMA (a vector encoding PUMA under thecontrol of the TATA-box sequence of the miR-21 promoter; SEQ ID NO: 29),together with the plasmid encoding Renilla luciferase.

FIG. 4B shows the results of co-transfection experiments performed asfor those depicted in FIG. 4A, except that a plasmid encoding GFP wasused as the reporter plasmid. Cells were harvested 72 hourspost-transfection and subjected to luciferase assay (FIG. 4A) or Westernblot analysis (FIG. 4B).

FIG. 5 demonstrates that miR-21 Pr DT-A expression inhibits tumorformation in vivo. Nude mice (n=3 per group) were injected with 1×10⁷HCT116 cells. Two weeks following tumor cell injection, mice wereadministered endotoxin-free miR-21 Pr DT-A (SEQ ID NO: 9) by intra-tumorinjection (dose of 25 ug). An additional injection was administered 7days later. Tumor size was recorded at days 0, 7 and 14 following tumorcell injection using a manual caliper. Control-treated mice wereadministered miR-21-Luc (SEQ ID NO: 11) by intra-tumor injection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to nucleic acid constructs and methodsfor providing targeted cancer therapy. Specifically, the constructs ofthe invention comprise at least one nucleic acid sequence encoding aheterologous anti-cancer gene product, the nucleic acid sequence beingoperably linked to a miR-21 transcription-regulating sequence, inparticular, an miR-21 promoter sequence. Vectors comprising theseconstructs, pharmaceutical compositions comprising them and methods ofuse thereof are also provided.

The efficacy of the present invention is unexpected over prior artdisclosures which teach suppression of miR-21 driven processes fortreatment of cancers in which miR-21 is highly expressed, such as byadministration of agents which inhibit miR-21 activity, or use of miR-21for inactivating genes involved in oncogenesis. That is, the harnessingof the miR-21 promoter for driving expression of tumor killing agents,as taught by the present invention, provides a reversal of prior artapproaches to therapy of miR-21-associated cancers.

Definitions

As used herein, the terms “microRNA” and “miRNA” interchangeably referto a single-stranded non-coding regulatory RNA of approximately 22-25nucleotides in length, generated by the action of RNase-III-type enzymeson an endogenous primary transcript (pri-miRNA).

As used herein, the terms “miRNA biogenesis”, “miRNA pathway” and “miRNAprocessing pathway” interchangeably refer to the RNA metabolic processleading to miRNA formation that includes transcription of the primarymiRNA transcript (pri-miRNA), cleavage of pri-miRNA to create anintermediate precursor miRNA (pre-miRNA) and subsequent processing ofpre-miRNA to create the mature miRNA.

As used herein, the term “miR-21” refers to the mature processed form ofthe miRNA having the sequence of SEQ ID NO: 5.

As used herein, the term “miR-21 promoter” refers to the native nucleicacid sequence which is located upstream of an miR-21 gene and isrequired for the transcription thereof, so as to produce a primarytranscript of miR-21 (pri-miR-21). Exemplary human pri-miR-21 sequencesinclude: GenBank accession number BC053563 (disclosed herein as SEQ IDNO: 2); GenBank accession number AY699265 (disclosed herein as SEQ IDNO: 3), and that disclosed in Fujita et al., J Mol Biol 2008 May 2;378(3):492-504 (disclosed herein as SEQ ID NO: 26). As used herein, theterm “miR-21 promoter sequence” refers to a nucleic acid sequencederived from an miR-21 promoter and having at least about 80% identityto SEQ ID NO:1.

As used herein, the term “an miR-21 transcription or processing product”refers to a nucleic acid transcription or processing product derivedfrom an miR-21 gene in the pathway to mature miR-21, including a primarytranscript of miR-21 (pri-miR-21), such as those having GenBankaccession numbers BC053563 and AY699265; an intermediate precursor ofmiR-21 (pre-miR-21), such as those having GenBank accession numbersNR_(—)029493, AF_(—)480546 and AF_(—)480557; and mature miR-21, such asthat having GenBank accession number NR_(—)029493.

As used herein, the term “nucleic acid sequence corresponding to orcomplementary to an miR-21 transcription or processing product” refersto a DNA or RNA sequence that is either identical to i.e. “correspondsto” the sequence of an miR-21 transcription or processing product, orcould demonstrate complete base-pairing i.e. is “complementary to” tothe miR-21 transcription or processing product. For example, a subjectDNA would “correspond to” a cDNA produced from an miR-21 RNA transcript,provided that the sequences of the subject DNA and the cDNA wereidentical, whereas the same subject DNA would be “complementary to” theaforementioned miR-21 transcript.

As used herein, the term “nucleic acid construct” refers to a continuouspolynucleotide molecule comprising at least two heterologous nucleicacid sequences that are joined together to form a single unit. Thenucleic acid sequences comprising the construct may include polypeptidecoding sequences and non-coding sequences.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid”,“nucleic acid sequence” and “oligonucleotide” are used interchangeablyherein to refer to polymeric forms of nucleotides of any length, such asdeoxyribonucleotides, ribonucleotides, or modified forms thereof in theform of an individual fragment or as a component of a larger construct,in a single strand or in a double strand or multi-strand form. Thepolynucleotides to be used in the invention include sense and antisensesequences of DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or apolymer comprising purine and pyrimidine bases or other natural,chemically or biochemically modified, non-natural, or derivatizednucleotide bases. Further included are mRNA or cDNA that compriseintronic sequences The backbone of the polynucleotide can comprisesugars and phosphate groups (as typically be found in RNA or DNA), ormodified or substituted sugar or phosphate groups. Alternatively, thebackbone of the polynucleotide can comprise a polymer of syntheticsubunits such as phosphoramidites and thus can be anoligodeoxynucleoside phosphoramidate or a mixedphosphoramidate-phosphodiester oligomer (see, e.g., Peyrottes et al.(1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi et al (1996) Nucl.Acids Res. 24:2318-2323). A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars, and linking groups such as fluororibose andthioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component, capping, substitution of one or more of naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support.

A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T) can also be uracil (U); this definition pertainsto the differences between the chemical structures of DNA and RNA, inparticular the observation that one of the four major bases in RNA isuracil (U) instead of thymidine (T).

The term “recombinant nucleic acid construct” as used herein refers to anucleic acid construct that has been subjected to molecular manipulationin vitro, such as for example joining together nucleic acid sequences byligation.

As used herein, the term “operably linked” refers to the structural andfunctional relationship between two or more nucleic acid sequencesjoined together as part of the same polynucleotide, generally aregulatory sequence and a protein coding sequence. For example, anucleic acid sequence encoding a protein is operably linked to apromoter if transcription is initiated from the promoter; or a nucleicacid sequence encoding a protein is operably linked to a ribosomebinding site if translation of the corresponding mRNA is initiated fromthe ribosome binding site. In the present invention, a nucleic acidencoding a protein having anti-cancer activity is operably linked to anmiR-21 transcription control sequence, preferably an miR-21 promotersequence, such that transcription of the nucleic acid is effected orenhanced. Generally, “operably linked” means that the nucleic acidsequences being linked are contiguous, although enhancers do not have tobe contiguous. Linking is accomplished by ligation at convenientrestriction sites, or if such sites do not exist, syntheticoligonucleotide adaptors or linkers may be used in accordance withconventional practice.

Transcription control sequences are sequences, which control theinitiation, elongation, and termination of transcription. Particularlyimportant transcription control sequences are those which controltranscription initiation, such as promoter, enhancer, operator andrepressor sequences.

As used herein, the term “vector” refers to a nucleic acid construct,comprising a regulatory sequence linked to a heterologous polynucleotidethat is inserted to target cells for replication and/or expressiontherein. The vector can be a viral expression vector, a plasmid or aconstruct of naked DNA, and, optionally, can include additionalsequences required for construction, selection, stability, penetration,etc.

As used herein, the terms “protein” and “polypeptide” referinterchangeably to a polymer of at least about 10 amino acids joinedtogether through peptide bonds. Throughout the specification, standardthree letter or single letter designations for amino acids are used.

As used herein, the terms “recombinant protein” and “recombinantpolypeptide” refer interchangeably to a protein produced using cellsthat do not have, in their native state, an endogenous copy of thenucleic acid (DNA or RNA) capable of expressing the protein. The cellsproduce the recombinant protein because they have been geneticallyaltered by the introduction of the appropriate isolated nucleic acidsequence, for example as a component of an expression vector.

As used herein, the term “fusion protein” as used herein refers to aprotein sequence composed of segments i.e. amino acid sequences, whichcorrespond to heterologous protein sequences, such as proteins ofdifferent function and/or proteins from different organisms. Thesegments are joined either directly or indirectly to each other viapeptide bonds, and may correspond to either full-length proteins orfragments thereof. By indirect joining it is meant that an interveningamino acid sequence, such as a peptide linker is juxtaposed betweensegments forming the fusion protein. Fusion proteins may be produced byrecombinant technology from a polynuceotide comprising the nucleic acidsequences encoding the various segments of the fusion protein.

As used herein, the term “drug-metabolizing enzyme” refers to an enzymewhich converts a prodrug into a cytotoxic product.

As used herein, the term “prodrug” refers to a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.

As used herein, the term “heterologous” when used in reference todistinct nucleic acid sequences or distinct protein sequences, meansthat the sequences differ with respect to their sequence, structureand/or the organism from they are derived or produced.

As used herein, the term “homolog” refers to a molecule, generally apolypeptide or nucleic acid sequence, which exhibits homology to anothermolecule, by having sequences of chemical residues that are identical orsimilar at corresponding positions.

Sequence similarity and sequence identity are calculated based on areference sequence, which may be a subset of a larger sequence, such asa conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. In general, percent sequence identity is calculated bycounting the number of residue matches (e.g., nucleotide residue oramino acid residue) between the query and test sequence and dividingtotal number of matches by the number of residues of the individualsequences found in the region of strongest alignment, or along acomplete region following the introduction of gaps, as is known in theart. Algorithms for computer-based alignment and sequence analysis areknown in the art (see, e.g., Altschul et al., (1990) J. Mol. Biol.,215:403-10), and publicly available software tools such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR), are well known in theart.

Accordingly, “percent (%) nucleic acid sequence identity” with respectto a miR-21 promoter nucleic acid sequence identified herein is definedas the percentage of nucleotides in a candidate sequence that areidentical with a disclosed miR-21 promoter nucleic acid sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved usingthe aforementioned computer software tools.

As used herein, the term “variant” refers to substantially similarsequences possessing common qualitative biological activities. Anoligonucleotide variant includes a pharmaceutically acceptable salt,homolog, analog, extension or fragment of a nucleotide sequence usefulfor the invention, such as an miR-21 promoter sequence. Encompassedwithin the term “variant” are chemically modified natural and syntheticnucleotide molecules. Also encompassed within the term “variant” aresubstitutions (conservative or non-conservative), additions or deletionswithin the nucleotide sequence of the molecule, as long as the requiredfunction is sufficiently maintained. Oligonucleotide and polynucleotidesvariants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity.

As used herein in the context of polynucleotides, the terms “hybridize”,“hybridizing”, “hybridizes” and the like, refer to conventionalhybridization conditions.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md., 1988.

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0. 1×SSC containing EDTA at 55° C.

As used herein, the term “isolated” in reference to a nucleic acidmolecule means that the nucleic acid molecule is initially separatedfrom different (in terms of sequence or structure) and unwanted nucleicacid molecules such that a population of isolated nucleic acids is atleast about 90% homogenous, and may be at least about 95, 96, 97, 98,99, or 100% homogenous with respect to other polynucleotide molecules.In many embodiments, a nucleic acid is isolated by virtue of it havingbeen synthesized in vitro separate from endogenous nucleic acids in acell. It will be understood, however, that isolated nucleic acids may besubsequently mixed or pooled together.

As used herein, the term “anti-cancer agent” refers to a protein orpeptide that exerts an inhibitory or cytotoxic effect on a cancer cell,either directly or indirectly. For example, a cytotoxic drug that killsa cancer cell exerts a direct effect on the cancer cell, while an enzymewhich converts a prodrug to its active cytotoxic form which then killsthe cancer cell, exerts an indirect effect.

The terms “cancer” and “neoplasm” are used herein interchangeably torefer to a disease state characterized by cells in an abnormal state orcondition characterized by rapid proliferation. A “tumor” containingsuch cells may be either benign, premalignant or malignant. The termsinclude disease states characterized by all types of hyperproliferativegrowth, hyperplastic growth, cancerous growths, oncogenic processes,metastatic tissues, malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness.

The term “carcinoma” is recognized by those skilled in the art andrefers to malignancies of epithelial or endocrine tissues includingrespiratory system carcinomas, gastrointestinal system carcinomas,genitourinary system carcinomas, testicular carcinomas, breastcarcinomas, prostatic carcinomas, endocrine system carcinomas, andmelanomas. Exemplary carcinomas include those forming from tissue of thecervix, lung, prostate, breast, head and neck, colon and ovary. The termalso includes carcinosarcomas, e.g., which include malignant tumorscomposed of carcinomatous and sarcomatous tissues. An “adenocarcinoma”refers to a carcinoma derived from glandular tissue or in which thetumor cells form recognizable glandular structures.

The term “sarcoma” is recognized by those skilled in the art and refersto malignant tumors of connective tissue such as bone or cartilage.

As used herein, the term “leukemia” and “leukemic cancer” refer to allcancers of the hematopoietic and immune systems (blood and lymphaticsystem). These terms refer to a progressive, malignant disease of theblood-forming organs, marked by distorted proliferation and developmentof leukocytes and their precursors in the blood and bone marrow.Myelomas refer to other types of tumors of the blood, bone marrow cells.Lymphomas refer to tumors of the lymph tissue.

As used herein, the term “pharmaceutical composition” refers to apreparation comprising one or more pharmaceutically active ingredientse.g. a nucleic acid construct encoding an anti-cancer agent, andgenerally further comprising at least one pharmaceutically acceptablediluent, carrier or excipient. The purpose of a pharmaceuticalcomposition is to facilitate administration of a pharmaceutically activeingredient to a subject.

As used herein, the term “active ingredient” refers to a component of apharmaceutical composition that provides the primary pharmaceuticalbenefit, as opposed to an “inactive ingredient” which is generallyrecognized as providing no pharmaceutical benefit.

As used herein, the term “carrier, excipient or diluent” refers to aninactive ingredient, for example a tonicity adjusting agent, wettingagent or preservative, which facilitates formulation and/oradministration of an active pharmaceutical ingredient.

As used herein, the term “pharmaceutically acceptable” refers to anon-toxic and inert substance that is physiologically compatible withhumans or other mammals.

As used herein, the term “subject” refers to any mammal of any agehaving a cancer, in particular a human subject, but also includingnon-human mammals, such as primate, canine, feline, bovine, equine andmurine species.

The term “antibody” (also referred to as an “immunoglobulin”) is used inthe broadest sense, and refers to polypeptides which exhibit bindingspecificity to a specific antigen. The term encompasses polyclonal andmonoclonal antibodies, including both full length antibodies andantibody fragments so long as they exhibit the desired biologicalactivity.

The terms “native” and “full length” antibodies” as used hereininterchangeably refer to heterotetrameric glycoproteins of about 150,000daltons, composed of two identical light (L) chains and two identicalheavy (H) chains. Each light chain is linked to a heavy chain bycovalent disulfide bond(s), while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain also has regularly spaced intrachaindisulfide bridges. Each heavy chain has, at one end, a variable domain(V_(H)) followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain.

Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to different classes. There arefive major classes of antibodies IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

The terms “polyclonal antibody” and “polyclonal antiserum” as usedherein refer to a population of antibody molecules synthesized by apopulation of B cells. Polyclonal antibodies encompass a population ofdifferent antibody molecules which specifically bind to a particularantigen, wherein various antibodies in the mixture recognize differentepitopes (also termed antigenic determinants).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, each monoclonal antibody is directed against asingle epitope on the antigen. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, monoclonal antibodies may be made by the hybridoma method firstdescribed by Kohler et al., Nature 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries, as is known in the art, for example using techniques such asthose described in Clackson et al., Nature 352:624-628 (1991) and Markset al., J. Mol. Biol. 222:581-597 (1991).

The terms “specifically interacts”, “specifically binds” and “havingspecificity for” are used herein interchangeably to refer to highavidity and/or high affinity binding of an antibody to an antigen orepitope thereof, e.g., an epitope on a cancer cell. Antibody binding toits epitope is stronger than binding of the same antibody to any otherepitope. Antibodies which bind specifically to a polypeptide of interestmay be capable of binding other polypeptides at a weak, yet detectable,level (e.g., 10% or less of the binding shown to the polypeptide ofinterest). Such weak binding, or background binding, is readilydiscernible from the specific antibody binding to the compound orpolypeptide of interest, e.g., by use of appropriate controls.

The singular forms “a”, “an”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a construct” includes one or more of such constructs, and so forth.

miR-21 Processing Pathway

Transcription of miRNA genes, including that of miR-21, is mediated byRNA polymerase II (pol II) to produce long primary transcripts(pri-miRNAs) that are often several kilobases long. Pri-miRNAtranscripts contain both a 5′ terminal cap structure and a 3′ terminalpoly(A) tail. Several poly(A)-containing transcripts containing bothmiRNA sequences and regions of adjacent mRNAs have been characterized. Ahuman pri-miR-21, 3463 nucleotides in length, is disclosed under GenBankaccession number AY699265, and a highly similar sequence from a humancDNA clone, 3404 nucleotides in length, is disclosed under GenBankaccession number BC053563. A human pri-miR-21 is disclosed by Fujita etal., J Mol Biol 2008 May 2; 378(3):492-504.

Homologous miR-21 genes of non-human species are also known, includingfor example those available under GenBank accession numbers AY865965(from Pan paniscus); AY865964 (from Ateles geoffroyi); AY865963 (fromMacaca nemestrina); AY865962 (from Pongo pygmaeus); AY865961 (fromGorilla gorilla); AY865960 (from Pan troglodytes), and AY865959 (fromMacaca mulatta).

The maturation of miRNA from pri-miRNAs involves trimming of pri-miRNAsinto hairpin intermediates called precursor miRNAs (pre-miRNAs), thatare subsequently cleaved into mature miRNAs. The stem-loop structure ofpri-miRNA molecules are cleaved by the nuclear RNase III enzyme Droshato release the pre-miRNA molecules. Drosha is a large protein ofapproximately 160 kDa, and, in humans, forms an even larger complex ofapproximately 650 kDa known as the Microprocessor complex. The enzyme isa Class II RNAse III enzyme having a double-stranded RNA binding domain(dsRBD).

Human sequences of pre-miR-21 include those available under GenBankaccession numbers NR_(—)29493; AF480546, and AF480557. Highly homologousnon-human pre-miR-21 sequences include those available under GenBankaccession numbers NR_(—)032094 (Equus caballus); NR_(—)030880 (Bostaurus); NR_(—)029738 (Mus musculus); and NR_(—)032151 (Monodelphisdomestica).

Following export of pre-miRNA molecules to the cytoplasm, another RNaseIII enzyme called “Dicer” cleaves the pre-miRNA to produce the maturemiRNA. Mature miRNAs are incorporated into an effector complex known asthe miRNA-containing RNA-induced silencing complex or miRISC.

Mature human miR-21 is available under GenBank accession numberNR_(—)29493 and is disclosed herein as SEQ ID NO:5. Identical or nearlyidentical non-human miR-21 sequences include those available underGenBank accession numbers NR_(—)032094 (Equus caballus); NR_(—)030880(Bos taurus); NR_(—)029738 (Mus musculus); and NR_(—)032151 (Monodelphisdomestica).

Additional information concerning miRNAs and associated pri-miRNA andpre-miRNA sequences is available in miRNA databases such as miRBase(Griffiths-Jones et al. 2008 Nucl Acids Res 36, (DatabaseIssue:D154-D158) and the NCBI human genome database.

It is to be noted that the sequences referred to and disclosed hereinare expressed with DNA thymine bases (T) rather than the correspondingRNA uracil (U) bases, even in cases where the native sequences are RNA,such as for example mature miR-21.

Nucleic Acid Constructs, Vectors and Host Cells

The nucleic acid construct of the invention comprises a nucleic acidsequence encoding an anti-cancer agent operably linked to an miR-21promoter sequence.

The miR-21 promoter sequence corresponds to a region positioned upstreamof an miR-21 gene that functions in transcription of the correspondingpri-miR-21 transcript. The miR-21 promoter sequence used for the presentinvention may be the native sequence or a fragment thereof, as long asit retains the capability to drive transcription of the nucleic acidsequence to which it is operably linked.

The miR-21 promoter for use in the invention may be that which drivestranscription of any of the known forms of pri-miR-21, such as thepri-miR-21 transcripts disclosed under GenBank accession numbersAY699265 and BC053563 and in Fujita et al., J Mol Biol 2008 May 2;378(3):492-504. Transcripts of miR-21 may be characterized by methodsknown in the art, for example rapid amplification of cDNA ends (RACE),polymerase chain reaction (PCR) and primer extension, as described forexample in Cai et al., RNA (2004) 10:1957-1966.

Putative promoter sequences located upstream i.e. 5′ of known oridentified pri-miR-21 transcription units may be PCR amplified fromgenomic DNA using appropriate primers, and isolated and purified usingstandard methods known in the art. Such purified fragments may then beligated into vectors comprising heterologous nucleic acid sequencesencoding reporter proteins, for example firefly luciferase. Such vectorsare then transfected into cells and analyzed for the presence of thereporter protein, thereby indicating whether the test sequence functionsas an mRNA promoter capable of driving expression of the reportersequence. In parallel systems, one or more shorter subfragments may beseparately cloned into the same vector and evaluated for reportersequence expression, so as to identify the minimal region with promoteractivity. A short subfragment, such as a putative TATA box, as well asantisense orientations of the various test sequences, may be similarlyprocessed to serve as negative controls.

Exemplary experiments of this type are disclosed in Example 2 herein,which shows that an miR-21 promoter sequence of SEQ ID NO: 1 drivesexpression of a luciferase reporter gene, while a short fragment of themiR-21 promoter corresponding only to the TATA box (SEQ ID NO: 6), isincapable of driving expression of the same reporter sequence.

Accordingly, an exemplary miR-21 promoter sequence for use in theinvention is SEQ ID NO: 1. SEQ ID NO 1 corresponds to nucleotides −1326to −608 relative to the pri-mir-21 T1 transcription start site disclosedin Fujita et al., J Mol Biol 2008 May 2; 378(3):492-504. In otherembodiments, the miR-21 promoter sequence has at least 80% identity withSEQ ID NO: 1, such as at least 85%, or at least 90% identity, or atleast 95% identity with SEQ ID NO: 1. In particular embodiments, themiR-21 promoter sequence comprises SEQ ID NO: 1.

In particular embodiments, the miR-21 promoter sequence comprisesrecognition sequences typically present in eukaryotic promoters, inparticular, the TATA box. Other promoter elements which may be presentin the miR-21 promoter sequence include initiator, CCAAT box and GC boxsites. The TATA box, typically located 25-30 base pairs upstream of thetranscription initiation site, is thought to be involved in directingRNA polymerase to begin RNA synthesis. The other upstream promoterelements determine the rate at which transcription is initiated.Preferably, the miR-21 promoter sequence utilized in the nucleic acidconstruct of the present invention is active in the specific target cellpopulation. In the constructs of the invention, it may be preferable toposition the miR-21 promoter sequence relative to the sequence encodingthe anti-cancer agent, such that the TATA box is the same distance fromthe transcription initiation site as occurs in the native gene encodingthe anti-cancer agent.

The nucleic acid sequence comprising an miR-21 promoter sequence may, insome embodiments, be substantially devoid of a nucleic acid sequencecorresponding to or complementary to an miR-21 transcription orprocessing product. furthermore, the nucleic acid construct may besubstantially devoid of a nucleic acid sequence corresponding to orcomplementary to a precursor or intermediate form of miR-21, such as aprimary transcript of miR-21 (pri-miR-21); a precursor of miR-21(pre-miR-21); an RNA duplex of miR-21, and a mature miR-21. Inparticular embodiments, the nucleic acid construct of the invention issubstantially devoid of a nucleic acid sequence corresponding to orcomplementary to a nucleic acid sequence selected from the groupconsisting of: SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; and SEQ ID NO:5.

In a particular embodiment, the nucleic acid construct comprises asequence selected from the group consisting of SEQ ID NO: 7 and SEQ IDNO: 8.

The constructs, vectors and nucleic acid sequences of the presentinvention may be produced using standard recombinant, enzymatic andchemical synthetic methods well known in the art. A combination of suchtechniques may be used. An isolated nucleic acid sequence can beobtained from its natural source, either as an entire (i.e., complete)gene or a portion thereof. The desired nucleic acid molecule may beproduced by polymerase chain reaction (PCR) amplification from a genomictemplate using suitable primer sequences. The amplified fragment may bemanipulated by addition of synthetic restriction endonuclease sites,linkers etc for cloning into a particular vector enabling propagationand/or further manipulation and/or expression of a particular nucleicacid sequence. Such procedures enable construction of a single moleculee.g. construct or vector, characterized by the juxtaposition of specificheterologous nucleic acid sequences and including suitable flankingsequences which enable the desired functionalities of the construct. Inaddition, at least some of the desired nucleic acid molecules may beproduced by assembly of chemically synthesized. oligonucleotides (seee.g. Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual;Ausubel et al., supra). For example, a nucleic acid sequence produced byoligonucleotide assembly may be ligated to a heterologous sequence thatwas produced by PCR amplification, and the resultant molecule may bemanipulated by addition of synthetic restriction endonuclease sites,linkers etc for cloning into a particular expression vector, as is wellknown in the art.

Nucleic acid sequences useful for the invention include natural nucleicacid sequences and homologs thereof, including, but not limited to,natural allelic variants and modified nucleic acid sequences in whichnucleotides have been inserted, deleted, substituted, and/or inverted insuch a manner that such modifications do not substantially interferewith the intended function of the nucleic acid such as encoding ananti-cancer agent or acting as a promoter for transcription thereof.

Any of the miR-21 promoter sequences used for the invention can bealtered by additions, substitutions or deletions and assayed for thelevel of expression of sequences operably linked thereto, in cancercells. Similarly, sequences encoding anti-cancer agents may be alteredin order to manipulate any of a number of properties, such as forexample the level of their toxicity, targeting capacity, in vivostability, etc.

Sequence alterations can be generated using a variety of chemical andenzymatic methods known to those skilled in the art. For example,regions of the sequences defined by restriction sites can be deleted.Oligonucleotide-directed mutagenesis can be employed to alter thesequence in a defined way and/or to introduce restriction sites inspecific regions within the sequence. Additionally, deletion mutants canbe generated using DNA nucleases such as Bal31 or ExoIII and S1nuclease. Progressively larger deletions in the regulatory sequences aregenerated by incubating the DNA with nucleases for longer periods oftime (See, e.g., Ausubel et al., supra for a review of mutagenesistechniques).

Sequence diversity can be introduced by a variety of mutagenesis schemesknown in the art. Random, non-specific methods of introducing mutationsinclude classical in vivo mutagenesis techniques, such as exposure ofentire organisms to radiation or chemical mutagens, or use oftransposons. More localized mutations in microorganisms in vivo can beperformed by using mutator strains of bacteria, transducing phages,episomes, or homologous recombination.

Mutations can be induced in vitro by many other methods in addition tohomologous recombination and random mutagenesis, which include deletionanalysis, linker mutations, reporter gene fusion, restriction/ligationassembly, oligonucleotide-directed mutagenesis, and cassettemutagenesis. PCR-based methods that can introduce sequence diversity arealso available such as error-prone PCR and assembly PCR (also known asparallel PCR). Furthermore, various combinations of the above techniqueshave led to specialized methods which include sexual PCR (also known asDNA shuffling), recursive ensemble mutagenesis and exponential ensemblemutagenesis.

Homologous recombination occurs naturally in living cells. Ineucaryotes, homologous recombination occurs at meiosis, and is thoughtto be one of the inherent evolutionary mechanisms. A number of patentsdescribe the use of in vivo homologous recombination to manipulate DNAsequences (see, e.g., U.S. Pat. Nos. 5,093,257; 5,413,923; 5,521,077;5,202,238; 5,763,240; and 6,015,708).

Means of performing random, or localized random, in vitro mutagenesisare essentially similar to those that can be used for in vivomutagenesis, e.g., irradiation, chemical mutagens, transposonmutagenesis. Aside from random mutagenesis methods, any of varioustechniques by which specific mutations can be made in vitro can beconsidered to be a form of site-directed mutagenesis (Kendrew, J., 1994,in The Encyclopedia of Molecular Biology, Blackwell Science Inc.,London). Non-PCR-based in vitro approaches to site-directed mutagenesiscan be grouped generally into the categories of oligonucleotide-directedmutagenesis, methods that restructure fragments of DNA (e.g., cassettemutagenesis, gene assembly), and localized random mutagenesis (Botsteinand Shortle, 1985, Science 229, 1193 1201).

Oligonucleotide-directed mutagenesis is based on the principle that anoligonucleotide encoding the desired mutation(s) is annealed to onestrand of the DNA of interest and serves as a primer for initiation ofDNA synthesis to produce a strand containing the mutation. Various formsof the basic technique, including single or multiple substitutions,insertions or deletions, are described by Kramer et al., 1982, NucleicAcids Res. 10:6475 6485; Zoller and Smith, 1982, Nucleic AcidsRes.10:6487 6500; Smith et al., 1982, “Site-Directed Mutagenesis”,Trends in Biochem. Sci., 7:440 442; and Norris et al., 1983, NucleicAcids Res. 11:5103 5112.

Cassette mutagenesis is characterized by replacement, typically byrestriction/ligation, of a portion of the endogenous gene with a“cassette”, which often is a synthetic oligonucleotide (see, e.g.,Estell et al., 1985, J. Biol. Chem. 260(11):6518 6521; Wells et al.,1985, Gene 34:315 323; Beck von Bodman et al., 1986, Proc Natl Acad Sci.83:9443 9447; Reidhaar-Olson and Sauer, 1988, Science 241:53 57; U.S.Pat. Nos. 4,894,331; 5,155,033; and 5,182,204).

Gene assembly entails the use of oligonucleotides, which can be amixture of synthetic oligomers and fragmented native sequences.Stochastic polymerization of the oligonucleotide pool by treatment witha DNA ligase results in the assembly of novel polynucleotide sequences(see, e.g. U.S. Pat. No. 5,723,323).

Error-prone PCR is a means of randomly introducing several pointmutations in a PCR product as a result of using a DNA polymerase thatdemonstrates low fidelity (see, e.g., Leung et al., 1989, Technique1:1115, 1989; U.S. Pat. No. 5,223,408).

Assembly PCR describes a process whereby, using a pool ofoligonucleotides, many different PCR reactions occur in parallel in thesame reaction mixture, with the products of one PCR reaction priming theproducts of another reaction (Stemmer et al., 1995, Gene 164:49-53). Themethod relies on DNA polymerase, rather than DNA ligase, to buildincreasingly longer DNA fragments during the assembly process (see e.g.,U.S. Pat. Nos. 5,605,793 and 5,830,696).

In sexual PCR, also called DNA shuffling, related but not identical DNAsequences are randomly fragmented, after which the fragments arereassembled by assembly PCR under conditions that permit homologousrecombination (Stemmer, 1994, Nature 370:389 391). Repeated cycles ofpoint mutagenesis, recombination, and selection produce in vitromolecular evolution. This process is repeated for as many cycles asnecessary to obtain a desired property or function (Stemmer, 1994, ProcNatl Acad Sci. 91:10747 10751).

Recursive Ensemble Mutagenesis (“REM”) is a protein engineering methodthat employs multiple cycles of cassette mutagenesis to identify“optimal” amino acids at targeted positions in a given protein. REM usesinformation gained from previous iterations of combinatorial cassettemutagenesis to search sequence space more efficiently. Through multiplerounds of optimized point mutation and recombination, rapid evolution ofDNA sequences is achieved (Arkin and Youvan, 1992, Proc Natl Acad Sci.89:7811 7815; Delagrave et al., 1993, Protein Engineering 6:327 33 1).

In exponential ensemble mutagenesis, several nucleotides are randomizedin parallel to identify amino acids, at each altered position, that leadto functional proteins. The method thereby generates combinatoriallibraries with a high percentage of optimized proteins. Exponentialensemble mutagenesis can be advantageous when it is desirable to changemany residues simultaneously. With the greater frequency of functionalmutants which is obtained by this method, entire proteins can bemutagenized combinatorially (Delagrave and Youvan, 1993, Biotechnology11:1548 1552; U.S. Pat. No. 5,521,077).

The altered sequences created by use of any technique known in the artto introduce sequence diversity, including those disclosed herein, areevaluated for their ability to direct expression of heterologouspolynucleotides in appropriate host cells, particularlymiR-21-expressing cancer derived cells e.g., pancreatic carcinoma cells.It is within the scope of the present invention to use any alteredmiR-21 promoter sequence that can direct tumor-specific expression tocreate a recombinant expression vector.

Synthetic methods suitable for preparing nucleic acid molecules includefor example, assembly of oligonucleotides (see e.g. U.S. Pat. No.5,583,013), or in vitro chemical synthesis using phosphotriester,phosphite or phosphoramidite chemistry and solid phase techniques suchas those described in EP 266,032, or via deoxynucleoside H-phosphonateintermediates (see e.g. U.S. Pat. No. 5,705,629). In the presentinvention, one or more oligonucleotides may be used to prepare thevarious nucleic acid molecules. Various different mechanisms ofoligonucleotide synthesis have been disclosed in for example, U.S. Pat.Nos. 4,659,774; 4,816,571; 5,141,813; 5,264,566; 4,959,463; 5,428,148;5,554,744; 5,574,146, and 5,602,244.

The nucleotide sequence encoding an anti-cancer agent can be selectedfrom those encoding a wide variety of proteins such as, but not limitedto, genes encoding toxic gene products, cytostatic gene products, geneproducts essential for viral replication, genes encoding drugmetabolizing enzymes and genes encoding inducers of apoptosis, asfurther described below.

The constructs of the invention may further comprise additionalheterologous protein-encoding sequences in addition to those encodingthe anti-cancer agent. These sequences may encode markers useful forselection or detection of cells transformed with the desired constructsin the course of cloning, propagation, manipulation, cell transfer,etc., such as, but not limited to, an enzyme or an antigenic marker.However, it is to be explicitly understood that the present inventionspecifically excludes constructs comprising an miR-21 promoter sequenceoperably linked to a nucleic acid sequence encoding a reporter protein,such as luciferase, as are known in the art.

Although the present invention relates to cell-specific expression ofheterologous polynucleotides to target cancer cells, greater specificityof treatment can be achieved by using cancer-specific delivery of thevectors of the invention. For example, antibodies that recognize cellsurface antigens unique to cancer cells, or that are more prevalent oncancer cells, compared to normal cells are known in the art, and can beused together with a vector of the invention to specifically target andkill tumor cells (See, e.g., Dillman, “Antibody Therapy: Principles ofCancer” Oldham (ed.), Raven Press, Ltd., New York, 1987). Accordingly,constructs according to the invention may include nucleic acid sequencesencoding antibodies or antigen binding fragments thereof that can serveto target the anti-cancer agent to the target cancer cell. In particularembodiments, the construct encodes a fusion protein, i.e. a singleprotein comprising heterologous protein segments, in which one segmentis the anti-cancer agent, and the other segment is an antibody orantibody fragment which is specific for a cancer-specific marker or acancer-related protein.

The nucleic acid constructs of the invention may be comprised in avector, in particular a eukaryotic expression vector. In particularembodiments, a vector useful for the invention may have the nucleic acidsequence of SEQ ID NO: 9 or SEQ ID NO: 10.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence or construct can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). The vectors ofthe present invention may include sequences which render the vectorsuitable for replication and integration in a particular type of hostcells, for example, prokaryotes or eukaryotes, or they may be capable ofpropagation in more than one type of host e.g. shuttle vectors which maybe propagated in both prokaryotes and eukaryotes.

Vectors suitable for cultivation of the subject polynucleotides inbacterial cells (e.g., E. coli), include, but are not limited to,plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.For replication in yeast, cloning and expression vehicles useful forintroducing genetic constructs in S. cerevisiae include, but are notlimited to, the YEP24, YIPS, YEP51, pYES2 and YRP17 plasmids (See, e.g.,Broach et al., 1993, in Experimental Manipulation of Gene Expression,ed. M. Inouye, Academic Press, p. 83). These vectors can replicate in E.coli due to the presence of the pBR322 ori, and in yeast due to thereplication determinant of the yeast 2 μm circle plasmid. In addition,drug-resistant markers such as ampicillin can be used.

Similarly, mammalian vectors for the polynucleotides of the inventionmay comprise prokaryotic sequences to facilitate the propagation of thevector in bacteria. Such vectors, when transfected into mammalian cells,can be designed to integrate into the mammalian chromosome for long-termstability using a linked selectable marker gene. Alternatively,derivatives of viruses such as the bovine papillomavirus (BPV-1) orEpstein-Barr virus can be used for transient expression

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and translation of an operably linked coding sequence in aparticular host organism. In addition to control sequences that governtranscription and translation, vectors and expression vectors maycontain nucleic acid sequences that serve other functions as well.

Control sequences typically found in expression vectors includetranscription and translation initiation sequences, transcription andtranslation terminators, and a polyadenylation signal.

Enhancer elements can augment transcription up to 1,000-fold from linkedhomologous or heterologous promoters. Enhancers are active when placeddownstream or upstream from the transcription initiation site. Manyenhancer elements derived from viruses have a broad host range and areactive in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus, human or murine cytomegalovirus (CMV), thelong term repeat from various retroviruses such as murine leukemiavirus, murine or Rous sarcoma virus and HIV. See, Enhancers andEukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. 1983.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase RNA stability. Two distinct sequence elements arerequired for accurate and efficient polyadenylation: GU or U richsequences located downstream from the polyadenylation site and a highlyconserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotidesupstream. Exemplary termination and polyadenylation signals that aresuitable for the present invention include those derived from SV40.

It is to be understood that while the miR-21 promoter sequence accordingto the invention may comprise at least some of the aforementionedelements, the construct or vector which comprises the miR-21 promotersequence may contain additional types of such elements. In addition tothe elements already described, the expression vector of the presentinvention may typically contain other specialized elements intended toincrease the level of expression of cloned nucleic acids or tofacilitate the identification of cells that carry the recombinant DNA.For example, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

In certain embodiments of the invention, the expression vector comprisesa virus or an engineered vector derived from a viral genome. The abilityof certain viruses to enter cells via receptor-mediated endocytosis, tointegrate into host cell genome and express viral genes stably andefficiently have made them attractive candidates for the transfer offoreign genes into mammalian cells (see e.g. Nicolas and Rubenstein,Biotechnology. 1988; 10:493-513; Baichwal and Sugden, Oncogene. 1988May; 2(5):461-7; Gélinas and Temin Proc Natl Acad Sci USA. 1986December; 83(23):9211-5; Coupar et al., Gene. 1988 Aug. 15; 68(1):1-10).Nucleic acid constructs can be inserted into a viral vector, such as,but not limited to, a papovavirus, a retrovirus, an adenovirus, anadeno-associated virus, a vaccinia virus or a herpesvirus (for review,see for example Viral Vectors: Gene Therapy and NeuroscienceApplications, Kaplitt and Loewy (eds.), Academic Press, San Diego(1995)).

The infection spectrum of viruses and viral-based vectors can be limitedby modifying the viral packaging proteins on the surface of the viralparticle (See, e.g., PCT publications WO93/25234 and WO94/06920).Strategies for modifying the infection spectrum of retroviral vectorsinclude, but are not limited to, coupling antibodies specific for cellsurface antigens to the viral env protein (Roux et al., 1989, Proc. Nat.Acad Sci. USA 86:9079 9083; Julan et al., 1992, J. Gen. Virol. u3:32513255; Goud et al., 1983, Virology 163:251 254) or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al., 1991, J. Biol.Chem 266:14143 14146). Coupling can be in the form of chemicalcrosslinking with a protein or other compound (e.g., lactose to convertthe env protein to an asialogycoprotein), or in the form of fusionproteins (e.g., single-chain antibody/env fusion proteins). Accordingly,cancer cells can be targeted to the surface of a recombinant virus byusing, for example, coupling antibodies that are directed againsttumor-associated molecules, or by using cancer cell surface proteins.Such techniques, while useful for limiting, or otherwise directing, theinfection to certain tissue types, can also be used to convert anectotropic vector into an amphotropic vector.

In a particular embodiment, an adenovirus-derived vector may be used inthe present invention. The genome of an adenovirus can be manipulatedsuch that it encodes and expresses a gene product of interest, but isinactivated in terms of its ability to replicate in a normal lytic virallife cycle (see, e.g., Berkner et al., 1988, BioTechniques 6:616;Rosenfeld et al., 1991, Science 252:431 434; Rosenfeld et al., 1992,Cell 68:143 155). Suitable adenoviral vectors derived from theadenovirus strain AD type 5 d1324 or other strains of adenovirus (e.g.,Ad2, Ad3, Ad7, etc.), are well known to those skilled in the art.

Recombinant adenoviruses can be advantageous in certain circumstances inthat they can be used to infect a wide variety of cell types, includingairway epithelium (Rosenfeld et al., 1992, Cell 68:143 155), endothelialcells (Lemarchand et al., 1992, Proc Natl Acad Sci. 89:6482 6486),hepatocytes (Herz and Gerard, 1993, Proc Natl Acad Sci. 90:2812 2816),and muscle cells (Quantin et al., 1992, Proc Natl Acad Sci. 89:25812584). Furthermore, the virus particle is relatively stable, amenable topurification and concentration, and can be modified so as to affect thespectrum of infectivity.

Additionally, introduced adenoviral DNA (and foreign DNA containedtherein) is generally not integrated into the genome of a host cell butremains episomal, thereby avoiding potential problems (e.g., insertionalmutagenesis) that can occur when introduced DNA (e.g., retroviral DNA)becomes integrated into the host genome.

Moreover, the carrying capacity of the adenoviral genome for foreign DNAis large (up to 8 kilobases) relative to other gene delivery vectors(Berkner et al., 1988, BioTechniques 6:616; Haj-Ahmand and Graham, 1986,J. Virol. 57:267). A replication-defective adenoviral vector useful forthe methods of the invention may have all or part of the viral E1 and E3genes deleted, but retains as much as 80% of the adenoviral geneticmaterial (see, e.g., Jones et al., 1979, Cell 16:683; Berkner et al.,supra; Graham et al. in Methods in Molecular Biology, Vol. 7, E. J.Murray (ed.), Humana, Clifton N.J. (1991) pp. 109-127).

Adeno-associated virus (AAV) can also be used in accordance with thepresent invention (see, e.g., Walsh et al., 1993, Proc. Soc. Exp. Biol.Med. 204:289-300; U.S. Pat. No. 5,436,146). Adeno-associated virus is anaturally occurring defective virus that requires another virus, such asan adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle (see, e.g., Muzyczka et al.,1992, Curr Top Microbiol Immunol. 158:97-129). Also, AAV is one of thefew viruses that can integrate its DNA into non-dividing cells, andexhibits a high frequency of stable integration (see, e.g., Flotte etal., 1992, Am J Respir Cell Mol Biol. 7:349 354; Samulski et al., 1989,J. Virol. 63:3822 3828; McLaughlin et al., 1989, J. Virol. 63:19631973).

Vectors containing as few as 300 base pairs of AAV can be packaged andcan integrate. The capacity for the insertion of exogenous DNA islimited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985, Mol. Cell. Biol. 5:3251-3260) can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see, e.g.Hermonat et al., 1984, Proc Natl Acad Sci. 81:6466-6470; 15 Tratschin etal., 1985, Mol. Cell. Biol. 4:2072-2081; Wondisford et al., 1988, Mol.Endocrinol. 2:32 39; Tratschin et al., 1984, J. Virol. 51:611-619;Flotte et al., 1993, J. Biol. Chem. 268:3781-3790).

Mammalian expression vectors which may serve as vector backbone for theconstructs of the present invention include, but are not limited to,pcDNA3, pcDNA3.1 (+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, and pNMT81, available from Invitrogen®; pCI available fromPromega®, pMbac, pPbac, pBK-RSV and pBK-CMV, available from Strategene®;and pTRES, available from Clontech®.

Other exemplary vectors include pSVT7 and pMT2 (derived from SV40);pBV-1MTHA (derived from bovine papilloma virus); pHEBO and p2O5;(derived from Epstein-Barr virus); pMSG; pAV009/A⁺; pMTO10/A⁺;pMAMneo-5, and baculovirus pDSVE.

Host cells transfected with vectors of the invention can be anyprokaryotic or eukaryotic cell. Transforming or transfecting the vectorinto host cells, either eukaryotic (e.g., yeast, avian, insect ormammalian) or prokaryotic (e.g., bacterial cells) are standardprocedures used widely in the microbial or tissue-culture technologies.

Methods suitable for nucleic acid delivery to effect expression ofanti-cancer agents according to the present invention include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnonviral vectors) can be introduced into an organelle, a cell, a tissueor an organism, as described herein or as would be known to one ofordinary skill in the art. Such methods include, but are not limited to,direct delivery of DNA such as by injection (see e.g. U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859), including microinjection (U.S. Pat.No. 5,789,215); by electroporation (U.S. Pat. No. 5,384,253); by calciumphosphate precipitation (Chen and Okayama, Mol. Cell Biol.,7(8):2745-2752, 1987); by using DEAE-dextran followed by polyethyleneglycol (Gopal, Mol. Cell Biol., 5:1188-1190, 1985); by direct sonicloading (Fechheimer et al., Proc. Natl. Acad Sci. USA, 84:8463-8467,1987); by liposome mediated transfection (Nicolau and Sene, Biochim.Biophys. Acta, 721:185-190, 1982; Kaneda et al., Science, 243:375-378,1989; Kato et al, J. Biol. Chem., 266:3361-3364, 1991); bymicroprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880; by agitation with silicon carbide fibers (U.S.Pat. Nos. 5,302,523 and 5,464,765); by Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,591,616 and 5,563,055); or byPEG-mediated transformation of protoplasts (U.S. Pat. Nos. 4,684,611 and4,952,500; by desiccation/inhibition-mediated DNA uptake (Potrykus etal., Mol. Gen. Genet., 199(2):169-77, 1985). Through the application oftechniques such as these, organelle(s), cell(s), tissue(s) ororganism(s) may be stably or transiently transformed.

Nucleic Acid Sequences Encoding Anti-Cancer Agents

The nucleotide sequence encoding an anti-cancer agent can be selectedfrom those encoding a wide variety of proteins such as, but not limitedto, cytotoxic gene products, cytostatic gene products, viral replicationproteins, drug metabolizing enzymes and inducers of apoptosis.

The anti-cancer agent may be a toxin, such as a bacterial toxin, a planttoxin, or a fungal toxin, either the full-length protein, but morepreferably a cytotoxic fragment of a naturally occurring toxin.Exemplary bacterial toxins include, but are not limited to, diphtheriatoxin, such as diphtheria toxin fragment A (DT-A), Pseudomonas exotoxin(PE), cholera toxin, anthrax toxin, botulinum toxin, pertussis toxin, E.coli enterotoxin, and shiga toxin. Exemplary plant toxins include, butare not limited to, ricin, modeccin, abrin, volkensin and viscumin.Examples of fungal toxins include α-sarcin (see for example, Nagano etal., J Antibiot (Tokyo). 1996 January; 49(1):81-5); restrictocin (seee.g., Rathore et al FEBS Lett 2 Sep. 1996, 392(3):259-262); mitogillin(see e.g., Better et al., J Biol Chem Aug. 15; 1992 267, 16712-16718);enomycin (see e.g., Takeuchi et al., J Antibiot (Tokyo). 1997 January;50(1):27-31); a fungal ribotoxin, for example RNase T1 (for review seee.g., Lacadena et al., FEMS Microbiol Rev. 2007 March; 31(2):212-37) andphenomycin (see e.g., Sakata et al., J Antibiot (Tokyo). 1994 March;47(3):370-1).

The toxin for use in the invention may be the A chain of a ricin-likeprotein, as disclosed for example in U.S. Pat. No. 7,375,186.

A diphtheria toxin for use in the invention may correspond to thefull-length 535 amino acid polypeptide secreted by Corynbacteriumdiphtheria or it may be a cytotoxic fragment thereof, in particularfragment A comprising the N-terminal catalytic domain (see, e.g.,Genbank Accession Nos. A04646 and AY820132; Greenfield et al., 1983Proc. Natl. Acad Sci. USA 80:6853-6857; Tweten et al., 1983 J.Bacteriol. 156:680-685; Kaczorek et al., 1983 Science 221:855-858; Leonget al., 1983 Science 220:515-517).

Examples 3 and 4 herein disclose a construct comprising a nucleic acidsequence encoding diphtheria toxin fragment A operably linked to amiR-21 promoter sequence and its cytotoxic effect on cancer cells invitro and in vivo.

In a particular embodiment, a nucleic acid sequence encoding adiphtheria toxin comprises SEQ ID NO: 18. In a particular embodiment,the construct encodes a diphtheria toxin comprising an amino acidsequence of SEQ ID NO: 19.

In other embodiments, a nucleic acid sequence encoding a Pseudomonasexotoxin (PE) may be used in the invention, for example thatcorresponding to the full-length protein of 613 amino acids secreted byPseudomonas aeruginosa, or that encoding a cytotoxic fragment thereof,such as a cytotoxic fragment corresponding to domain III i.e. amino acidresidues 400-613 (see, e.g., U.S. Pat. No. 5,602,095; Siegall et al., J.Biol. Chem. 264:14256-14261, 1989). Other forms of PE which may beencoded and expressed by the constructs of the invention include forexample, PE38 (see e.g., U.S. Pat. No. 5,608,039, and Pastan et al.,1997, Biochim. Biophys. Acta 1333:C1-C6), disclosed herein as SEQ ID NO:21; PE38 KDEL (see e.g., Brinkmann et al 1991, Proc Nat Acad Sci USA88:8616-8621), disclosed herein as SEQ ID NO: 22; PE37 (see e.g., U.S.Pat. No. 5,602,095) disclosed herein as SEQ ID NO: 23; and PE40 (seee.g., U.S. Pat. No. 6,051,405) disclosed herein as SEQ ID NO: 24.

The invention further encompasses use of a nucleic acid encoding adrug-metabolizing enzyme which converts a prodrug into a cytotoxicproduct.

In a particular embodiment, the drug-converting enzyme is a thymidinekinase (e.g. from herpes simplex virus or varicella zoster virus), whichhas the capacity to convert the prodrug form of ganciclovir(2-amino-9-{[(1,3-dihydroxypropan-2-yl)oxy]methyl}-6,9-dihydro-3H-purin-6-one)into its active phosphorylated form, which is a deoxyguanosinetriphosphate (dGTP) analog. This analog competitively inhibits theincorporation of dGTP into cellular DNA, resulting in cell death.

Drug metabolizing enzymes that convert a prodrug into a cytotoxicproduct include, but are not limited to, thymidine kinase (e.g. fromherpes simplex virus or varicella zoster virus), cytosine deaminase,nitroreductase, cytochrome p-450 2B1, thymidine phosphorylase, E. coliguanine phosphoribosyl transferase, purine nucleoside phosphorylase,alkaline phosphatase, carboxypeptidases A and G2, linamarase,beta-lactamase, xanthine oxidase, and variants thereof (see, e.g.,Pandha et al., 1999, J Clin Oncol. 17:2180 2189; Rigg and Sikora, 1997,Mol Med Today. 3:359 366).

The anti-cancer agent may further be an inducer of apoptosis, such asPUMA (see, e.g. GenBank accession No. AF354654); BAX (see, e.g. GenBankaccession No. AY217036); BAK (see, e.g. GenBank accession No.NR_(—)027882); BcI-XS (see, e.g. GenBank accession No. NM_(—)001191);BAD (see, e.g. GenBank accession No. BCO01901); BIM (see, e.g. GenBankaccession No. BC033694); BIK (see, e.g. GenBank accession No. U34584);BID (see, e.g. GenBank accession No. BC036364); HRK (see, e.g. GenBankaccession No. HSU76376); Ad E1B; ICE-CED3 proteases (see, e.g. GenBankaccession Nos. NM_(—)001080124 and NP_(—)001073593); TRAIL (see, e.g.GenBank accession Nos. NM_(—)003810 and NP_(—)003801); SARP-2 (see, e.g.GenBank accession Nos. NM_(—)003012 and NP_(—)003003); and apoptin (see,e.g. GenBank accession Nos. NM_(—)012234 and NP_(—)036366).

Oligonucleotide sequences useful for preparing nucleic acid sequencesencoding such apoptosis inducers are disclosed for example in U.S.Patent Application publication No. 2009/0298071.

Examples 3 and 4 herein discloses construction and use of a constructand vector comprising a sequence encoding PUMA for use in anti-cancertherapy.

Polynucleotides encoding cytostatic agents that suppress cell growth andmultiplication are useful for the invention. These include, but are notlimited to, gene products derived from p21 (Waf1), p27 (Kip1), p53,p53175P, p57 (Kip2), p15 (INK4b), p16 (INK4a), p18(INK4c), p19(Arf),p73, GADD45, APC1, p73RB1, WT1, NF1, VHL, and variants thereof (seee.g., Koga et al., Hepatology. 33:1087 1097; Huang et al., 2001, CancerRes. 61:3373 3381; Li et al., 2001, Cancer Res. 61:1493 1499; Mazur etal., 1998, Biotechnol Prog. 14:705 713; Tsugu et al., 2000, Am J Pathol.157:919 932; Dyer et al., 2001, J Neurosci. 21:4259 4571; Kovalev etal., 2001, J Immunol. 167:3285 3292; Modesitt et al., 2001, Clin CancerRes. 7:1765 1772; Wong et al., 2001, J Pathol. 194:35 42; Shapiro etal., 2000, Cell Biochem Biophys. 33:189 197; Fuxe et al., 2000, CellGrowth Differ. 11:373 384; Latres et al., 2000, EMBO J. 19:3496 3506;Weber et al., 2000, Genes Dev. 14:2358 2365; Sasaki et al., 2001, GeneTher. 8:1401 1408; Zhu et al., 1998, Cancer Res. 58:5061 5065; Mullan etal., 2001, Oncogene. 20:6123 6131; Velasco-Miguel et al., 1999,Oncogene. 18:127 137; Jorgensen et al., 2001, Gene. 262:51 59; Kurasawaand Todokoro, 1999, Oncogene 18:5131 5137; Basu et al., 1999, Int J.Oncol. 15:701 708; Yamagami et al., 1998, Leuk Res. 22:383 384; Murataet al., 1997, FEBS Lett. 409:41 45; Uhlmann et al., 2001, Cell BiochemBiophys. 34:61 78; Zhang et al., 1998, J Exp Med. 187:1893 1902; Nortonet al., 1996, Neuroreport. 7:601 604; Baba et al., 2001, Oncogene.20:2727 2736; Davidowitz et al., 2001, Mol Cell Biol. 21:865 874).

Pharmaceutical Compositions, Formulations and Modes of Administration

The nucleic acid constructs and vectors of the invention may beadministered to a subject alone or in the form of a pharmaceuticalcomposition. Pharmaceutical compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the nucleic acids into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, inhalation, oral or pulmonary administration. Forinjection, the nucleic acids of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the nucleic acidmolecules may be in powder form for reconstitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. For administration by inhalation, themolecules for use according to the present invention are convenientlydelivered in the form of an aerosol spray from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the nucleic acids and a suitable powder basesuch as lactose or starch. The nucleic acid molecules may also beformulated in rectal or vaginal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides. For oral administration, thenucleic acids can be readily formulated by combining the molecules withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the nucleic acids of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, e.g.lactose, sucrose, mannitol and sorbitol; cellulose preparations such asmaize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulatingagents; and binding agents. If desired, disintegrating agents may beadded, such as the cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. If desired, solid dosageforms may be sugar-coated or enteric-coated using standard techniques.For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added. For buccaladministration, the molecules may take the form of tablets, lozenges,etc. formulated in conventional manner.

The nucleic acid molecules may also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the molecules may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

A nucleic acid of the invention may be administered in combination witha carrier or lipid to increase cellular uptake. For example, nucleicacids may be administered in combination with a cationic lipid. Examplesof cationic lipids include, but are not limited to, Lipofectin®, DOTMA,DOPE, and DOTAP, and T-shaped cholesterol ester derivative (see e.g. Leeet al Biorg Med Chem Lett 14 (2004):2637-2641. Various nucleic acidformulations and methods of administration are disclosed for example inU.S. Pat. Nos. 7,470,675; 5,844,107; 5,877,302; 6,008,336; 6,077,835;6,200,801, and 5,972,900, and in U.S. Patent Application PublicationNos. 2003/0203865; 2002/0150626; 2003/0032615, and 2004/0048787.

In addition, liposomes and emulsions are well-known examples of deliveryvehicles that may be used to deliver nucleic acids of the invention.Suitable liposomes can be formed from standard vesicle-forming lipids,which generally include neutral or negatively charged phospholipids anda sterol, such as cholesterol. The selection of lipids is generallyguided by consideration of factors, such as the desired liposome sizeand half-life of the liposomes in the blood stream. A variety of methodsare known for preparing liposomes, described example, in Szoka et al.(1980), Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369.

Liposomes for use in the present methods can comprise a ligand moleculethat targets the liposome to cancer cells, for example a monoclonalantibody that binds a tumor cell antigen.

can also be modified so as to avoid clearance by the mononuclearmacrophage system (“MMS”) and reticuloendothelial system (“RES”). Suchmodified liposomes have opsonization-inhibition moieties on the surfaceor incorporated into the liposome structure. In a particular embodiment,a liposome of the invention can comprise both an opsonization-inhibitionmoiety and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization-inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016.

Examples of opsonization-inhibiting moieties suitable for modifyingliposomes are water-soluble polymers with a number-average molecularweight from about 500 to about 40,000 daltons, such as about 2,000 toabout 20,000 daltons. Such polymers include polyethylene glycol (PEG) orpolypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, andPEG or PPG stearate; synthetic polymers, such as polyacrylamide or polyN-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines;polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitolto which carboxylic or amino groups are chemically linked, as well asgangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG,or methoxy PPG, or derivatives thereof, are also suitable. In addition,the opsonization-inhibiting polymer can be a block copolymer of PEG andeither a polyamino acid, polysaccharide, polyamidoamine,polyethyleneamine, or polynucleotide. The opsonization-inhibitingpolymers can also be natural polysaccharides containing amino acids orcarboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronicacid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid,carrageenan; aminated polysaccharides or oligosaccharides (linear orbranched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups. Preferably, the opsonization-inhibiting moiety is aPEG, PPG, or a derivative thereof Liposomes modified with PEG orPEG-derivatives are sometimes called “PEGylated liposomes.”

An opsonization-inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects, for example, solid tumors, will efficiently accumulate theseliposomes; see Gabizon, et al. (1988), Proc. Natl. Acad Sci., U.S.A.,18:6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation ofthe liposomes in the liver and spleen. Thus, liposomes that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe or nucleic acids of the invention to tumor cells.

The nucleic acids may also be administered in combination with acationic amine such as poly (L-lysine). Nucleic acids may also beconjugated to a chemical moiety, such as transferrin and cholesteryls.In addition, oligonucleotides may be targeted to certain organelles bylinking specific chemical groups to the oligonucleotide. For example,linking the oligonucleotide to a suitable array of mannose residues willtarget the oligonucleotide to the liver.

Additionally, the molecules may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the molecules for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the chimericmolecules, additional strategies for molecule stabilization may beemployed.

Nucleic acids may be included in any of the above-described formulationsas the free acids or bases or as pharmaceutically acceptable salts.Pharmaceutically acceptable salts are those salts that substantiallyretain the biologic activity of the free bases and which are prepared byreaction with inorganic acids. Pharmaceutical salts tend to be moresoluble in aqueous and other protic solvents than are the correspondingfree base forms.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more nucleic acid molecules or vectorsdissolved or dispersed in a pharmaceutically acceptable carrier. Thephrases “pharmaceutically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one nucleic acid ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by health regulatoryagencies such as the FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier, diluent orexcipient” includes any and all solvents, dispersion media, coatings,surfactants, antioxidants, preservatives (e.g., antibacterial agents,antifungal agents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art. Except insofar as any conventionalcarrier is incompatible with the active ingredient, its use in thetherapeutic or pharmaceutical compositions is contemplated.

The pharmaceutical compositions comprising nucleic molecules maycomprise different types of carriers depending on whether it is to beadministered in solid, liquid or aerosol form, and in accordance withthe general need for sterility for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, inhalation (e.g. aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art.

The actual dosage amount of a composition of the present inventionadministered to the subject, such as a human patient, can be determinedby physical and physiological factors such as body weight, severity ofcondition, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from at least about 1microgram/kg/body weight, including about 5, about 10, about 50, about100, about 200, about 350 weight, about 500 microgram/kg/body weight; orat least about 1 milligram (mg)/kg/body weight, such as about 5, about10, about 50, about 100, about 200, about 350, about 500, to about 1000mg/kg/body weight or more per administration, and any range derivabletherein.

The composition may comprise various antioxidants to retard oxidation ofone or more component. Additionally, the composition may comprisepreservatives such as various antibacterial and antifungal agents,including but not limited to parabens (e.g., methylparabens,propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal orcombinations thereof.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use nasal solutions or sprays, aerosols orinhalants in the present invention. Such compositions are generallydesigned to be compatible with the target tissue type. In a non-limitingexample, nasal solutions are usually aqueous solutions designed to beadministered to the nasal passages in drops or sprays. Nasal solutionsare prepared so that they are similar in many respects to nasalsecretions, so that normal ciliary action is maintained. Thus, inpreferred embodiments the aqueous nasal solutions usually are isotonicor slightly buffered to maintain a pH of about 5.5 to about 6.5.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

Therapeutic Uses and Methods of Treatment

The nucleic acid constructs and vectors of the invention may be used fortreating a subject having cancer, such as for inhibiting tumorprogression, for inhibiting tumor metastasis, or for reducing oralleviating a symptom associated with a neoplastic disorder.

The invention is particularly useful for treating types of cancer,tumors or neoplastic disorders characterized inter alia by endogenousexpression of miR-21 in at least a portion of the cells thereof.

The cancer, tumor or neoplastic disorder may be a sarcoma, a carcinoma,an adenocarcinoma, a lymphoma, and a leukemia. Included within the scopeof the invention are breast cancer, colon cancer, hepatocellularcarcinoma, cervical cancer, cholangiocarcinoma, endometrioid ovariancarcinoma, esophageal cancer, glioblastoma, head and neck cancer,leukemia (e.g. chronic lymphocytic leukemia), lymphoma (e.g. diffuselarge B cell lymphoma, activated B cell-like lymphoma), lung cancer,multiple myeloma, pancreatic (e.g. endocrine and acinar) cancer,osteosarcoma, pituitary tumor, prostate cancer, stomach cancer, anduterine leiomyoma.

The constructs and vectors of the invention treat cancer by inhibitingcancer cell proliferation, and/or by arresting or slowing the growth ofthe cancer cells and/or by inhibiting tumor progression, and/or byinhibiting tumor metastasis. Any of the aforementioned effects may bepermanent or temporary. Inhibition of cancer cell proliferation can beinferred if the number of such cells in the subject remains constant ordecreases after administration of the constructs and vectors. Aninhibition of cancer cell proliferation can also be inferred if theabsolute number of such cells increases, but the rate of tumor growthdecreases.

The number of cancer cells in the body of a subject can be determined bydirect measurement, or by estimation from the size of primary ormetastatic tumor masses. For example, the number of cancer cells in asubject can be measured by immunohistological methods, flow cytometry,or other techniques designed to detect characteristic surface markers ofcancer cells.

The size of a tumor mass can be ascertained by direct visualobservation, or by diagnostic imaging methods, such as X-ray, magneticresonance imaging, ultrasound, and scintigraphy. Diagnostic imagingmethods used to ascertain size of the tumor mass can be employed with orwithout contrast agents, as is known in the art. The size of a tumormass can also be ascertained by physical means, such as palpation of thetissue mass or measurement of the tissue mass with a measuringinstrument, such as a caliper.

Any mode of administration of the constructs and vectors can be used solong as it results in the expression of the anti-cancer agent in thedesired tissue, in an amount sufficient to be therapeutically and/orprophylactically effective. The miR-21 promoter containing constructsand vectors can be administered to a subject by any suitable enteral orparenteral administration route. Suitable enteral administration routesfor the present methods include, e.g., oral, rectal, or intranasaldelivery. Suitable parenteral administration routes include, e.g.,intravascular administration (e.g., intravenous bolus injection,intravenous infusion, intra-arterial bolus injection, intra-arterialinfusion and catheter instillation into the vasculature); peri- andintra-tissue injection (e.g., peri-tumoral and intra-tumoral injection,intra-retinal injection, or subretinal injection); subcutaneousinjection or deposition, including subcutaneous infusion (such as byosmotic pumps); direct application to the tissue of interest, forexample by a catheter or other placement device (e.g., a retinal pelletor a suppository or an implant comprising a porous, non-porous, orgelatinous material e.g. gelfoam sponge depots; hydrogels) or topicalapplications during surgery; and inhalation. Particularly suitableadministration routes are injection, infusion and direct injection intothe tumor.

Means of injection include biolistic injectors and particle accelerators(e.g., “gene guns” or pneumatic “needleless” injectors), such as thosemarketed as Med-E-Jet™ (Vahlsing, H., et al., J. Immunol. Methods 171,11-22 (1994)), Biojector™ (Davis, H., et al., Vaccine 12, 1503-1509(1994); Gramzinski, R., et al., Mol. Med. 4, 109-118 (1998)),AdvantaJet™ (Linmayer, I., et al., Diabetes Care 9:294-297 (1986)), andMedi-Jector™ (Martins, J., and Roedl, E. J. Occup. Med. 21:821-824(1979)).

The nucleic acid constructs and vectors may be administered as a singledose or multiple doses, administered at suitable time intervals.

The term “therapeutically effective amount” refers to an amount of aconstruct or vector of the invention that is effective to achieve thedesired result following administration.

The constructs and vectors may be administered in conjunction with oneor more additional modalities of cancer therapy including, but notlimited to bone marrow transplant, cord blood cell transplant, surgery,chemotherapy, radiation therapy, and immunotherapy. The polynucleotideconstruct or pharmaceutical composition of the present invention can beadministered prior to the commencement of one or more of the additionalcancer therapies, during the practice of one or more of the additionalmodalities of cancer therapy, and after the end of one or more of theadditional modalities of cancer therapy.

Types of bone marrow transplant include, but are not limited toautologous bone marrow transplant and heterologous (i.e., from a donor)bone marrow transplant.

Chemotherapeutic agents include, but are not limited to alkylatingagents, including mechlorethamine, cyclophosphamide, ifosfamide,melphalan, chlorambucil, dicarbazine, streptazocine, carmustine,lomustine, semustine, chlorozotocin, busulfan, triethylenemelamine,thiotepa, hexamethylmelamine; antimetabolites, including methotrexate;pyrimidine analogs, including fluorouracil, 5-fluorouracil, floxuridine(5′-fluoro-2′-deoxyuridine), idoxuridine, cytarabine,N-phosphonoacetyl-L-aspartate, 5-azacytidine, azaribine, 6-azauridine,pyrazofuran, 3-deazauridine, acivicin; purine analogs, includingthioguanine, mercaptopurine, azathioprine, pentostatin,erythrohydroxynonyladenine; vinca alkaloids, including vincristine andvinblastine; epipodophyllotoxins, including etoposide and teniposide;antibiotics, including dactinomycin, daunorubicin, doxorubicin,bleomycin sulfate, plicamycin, mitomycin; enzymes, includingL-asparaginase; platinum coordination complexes, including cisplatin,carboplatin; hydroxyurea, procarbazine, mitotane; and hormones orrelated agents, including adrenocorticosteroids such as prednisone andprednisolone; aminoglutethimide; progestins such as hydroxyprogesteronecaproate, medroxyprogesterone acetate, megesterol acetate, estrogens andandrogens such as diethylstilbestrol, fluoxymesterone, ethynylestradiol, antiestrogens such as tamoxifen, and gonadotropin-releasinghormone analogs such as leuprolide.

The methods of the invention may further comprise a step of determiningthe level of miR-21 transcriptional activity in a biological sample e.g.cells or tissue, from the subject. The presence or level of at least onemiR-21 gene product in the sample may be indicative that the cancer ortumor or neoplasm is treatable by the methods of the invention.

The level of a miR-21 gene product can be measured in a biologicalsample obtained from the subject. For example, a tissue sample can beremoved from a subject suspected of having breast cancer by conventionalbiopsy techniques. In another example, a blood sample can be removedfrom the subject, and white blood cells can be isolated for DNAextraction by standard techniques. The blood or tissue sample ispreferably obtained from the subject prior to initiation of treatmentaccording to the invention or any other type of intervention such asradiotherapy, chemotherapy or other therapeutic treatment. Acorresponding control tissue or blood sample can be obtained fromunaffected tissues of the subject, from a normal human individual orpopulation of normal individuals, or from cultured cells correspondingto the majority of cells in the subject's sample. The control tissue orblood sample is then processed along with the sample from the subject,so that the levels of miR-21 gene product produced from a miR-21 gene incells from the subject's sample can be compared to the correspondingmiR-21 gene product levels from cells of the control sample.

Some cancers are characterized by an alteration, particularly anincrease, in the level of an miR-21 gene product in a sample obtainedfrom the subject, relative to the level of a corresponding miR-21 geneproduct in a control sample. When the level of at least one miR-21 geneproduct in the test sample is greater than the level of thecorresponding miR-21 gene product in the control sample, the expressionof the miR-21 gene product is “up-regulated”.

The relative miR-21 gene expression in the control and normal samplescan be determined with respect to one or more RNA expression standards.The standards can comprise, for example, a zero miR-21 gene expressionlevel, the miR-21 gene expression level in a standard cell line, or theaverage level of miR-21 gene expression previously obtained for apopulation of normal human controls.

The level of a miR-21 gene product in a sample can be measured using anytechnique that is suitable for detecting RNA expression levels in abiological sample. Suitable techniques for determining RNA expressionlevels in cells from a biological sample (e.g., Northern blot analysis,RT-PCR, in situ hybridization) are well known to those of skill in theart. In a particular embodiment, the level of at least one miR-21 geneproduct is detected using Northern blot analysis. For example, totalcellular RNA can be purified from cells by homogenization in thepresence of nucleic acid extraction buffer, followed by centrifugation.Nucleic acids are precipitated, and DNA is removed by treatment withDNase and precipitation. The RNA molecules are then separated by gelelectrophoresis on agarose gels according to standard techniques, andtransferred to nitrocellulose filters. The RNA is then immobilized onthe filters by heating. Detection and quantification of specific RNA isaccomplished using appropriately labeled DNA or RNA probes complementaryto the RNA in question. See, for example, Sambrook et al., supra Chapter7.

Suitable probes for Northern blot hybridization of a given miR-21 geneproduct can be produced from their known nucleic acid sequences, asdisclosed herein and in publicly available databases. Methods forpreparation of labeled DNA and RNA probes, and the conditions forhybridization thereof to target nucleotide sequences, are described inSambrook et al., supra.

Probes can be labeled for example with a radionuclide, a heavy metal; aligand capable of functioning as a specific binding pair member for alabeled ligand (e.g., biotin, avidin or an antibody), a fluorescentmolecule, a chemiluminescent molecule, an enzyme or the like, to highspecific activity using methods well known in the art. AutoradiographicDetection of hybridization is performed by a means appropriate for thelabeling entity e.g. autoradiography for radiolabeled probes; reactingbiotinylated probes with biotin-binding proteins that produce colorreactions.

In situ hybridization involves depositing whole cells onto a microscopecover slip and probing the nucleic acid content of the cell with asolution containing radioactive or otherwise labeled nucleic acidprobes. This technique is particularly well-suited for analyzing tissuebiopsy samples from subjects (see e.g. U.S. Pat. No. 5,427,916).

The relative number of miR-21 gene transcripts in cells can also bedetermined by reverse transcription of miR-21 gene transcripts, followedby amplification of the reverse-transcribed transcripts by polymerasechain reaction (RT-PCR). The levels of miR-21 gene transcripts can bequantified in comparison with an internal standard, for example, thelevel of mRNA from a “housekeeping” gene present in the same sample. Asuitable “housekeeping” gene for use as an internal standard includes,e.g., myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Themethods for quantitative RT-PCR and variations thereof are within theskill in the art.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miRNA gene products in asample. In other instances, it may be desirable to determine theexpression level of the transcripts of all known miRNA genes correlatedwith a cancer. Suitable microarray techniques are known in the art, asdescribed for example in U.S. Pat. Application Publication No.2008/0306018.

Kits

The present invention also provides kits containing compositions of theinvention or compositions to implement methods of the invention. In someembodiments, the kits are for therapeutic use and contain multipledosage units of the constructs or vectors and instructions for use foradministering to a subject having cancer.

In some embodiments, the kits are for prognostic use, for example toevaluate the susceptibility of one or more miR-21 expressing targetcells to the cytotoxic effects of the construct of the invention.

Individual components of the kit may be provided in concentratedamounts; in some embodiments, a component is provided individually inthe same concentration as it would be in a solution with othercomponents.

Negative and/or positive control vectors are included in some kitembodiments. The control molecules can be used to verify transfectionefficiency and/or control for transfection-induced changes in cells. Thekit may also include one or more transfection reagent(s) to facilitatedelivery of the construct or vector to cells.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the nucleic acids, and any other reagent containers inclose confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent, provided ina separate container means.

The kits may also include components that preserve or maintain theconstructs and vectors or that protect against its degradation.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention.

EXAMPLES Example 1 Differential Expression of Endogenous miR-21 inCancer Cells and tissues.

Table 1 summarizes the differential miR-21 expression in cancerous vs.non-cancerous tissues and cells (NS=not specified).

TABLE 1 Fold over expression Tumor/cancer type Method (Mean) ReferenceGlioblastoma Microarray, Northern 10-40  [5] blots  [6] real-time PCRBreast cancer Microarray 1.6  [7] In situ hybridization NS  [8]Real-time PCR 5  [9] Real-time PCR NS [10] Microarray 2 [11]Hepatocellular Microarray ~2 [12] carcinomas small RNA cloning 4.5 [13]and sequencing real-time PCR >2 [14] Microarray, real-time 10-45 [15]PCR Cholangiocarcinoma Northern blots and  6-12 [16] real-timepolymerase chain reaction Pancreatic endocrine Microarray NS [17] andacinar tumors Pancreatic cancer Microarray 15.7 [18] In situhybridization NS [19] Lung, breast, stomach, Microarray NS [20]prostate, colon, and pancreatic tumors Chronic lymphocytic miRNA cloningand ~3 [21] leukemia real-time-PCR Osteosarcoma miRNA cloning and NS[22] real-time-PCR, Northern blots Endometrioid ovarian Microarray,Northern 4 [23] carcinoma blots Diffuse large B cell Microarray 20 [24]lymphoma (activated B [25] cell-like) Cervical cancer Direct sequencing5 [26] Multiple myeloma NS NS [27] Microarray, Real-time- [28] PCR Coloncancer Real-time-PCR 5 [29] Microarray NS [30] Microarray NS [31]Real-time-PCR 10-70 [32] Head and neck cancer Microarray, Northern NS[33] blots Esophageal cancer Microarray 7.2 [34] Microarray 3-5 [35]Uterine leiomyomas Microarray 1.2-5   [36] Pituitary tumorsReal-time-PCR 2.4 [37] Lung cancer Real time RT-PCR NS [38] Ovariancarcinoma Microarray, Northern 3 [39] blots Bladder cancer Microarray 10[40]

Example 2 Expression of Heterologous Genes Under miR-21 Promoter Control

A recombinant vector that expresses the firefly luciferase gene (Luc)under the control of a miR-21 promoter sequence (miR-21-Luc; SEQ ID NO:11) was produced by standard recombinant procedures, as detailed herein.As a negative control, a vector containing the TATA box of the miR-21promoter (TATA-Luc; SEQ ID NO: 20), rather than the miR-21 promoter, wasprepared.

The human hepatocellular carcinoma cell line Huh7 was used as a genomicDNA template for amplification of an miR-21 promoter sequence of SEQ IDNO: 1, using the following primers: fw 5′-GGGGTACCGAAGGAGCTCCGAGTACATAAAT-3′ (SEQ ID NO: 14) and rev 5′-CCCAAGCTTCTACTCTGGTATGGCACAAAG 3′(SEQ ID NO: 15). The PCR product was cloned into the pGL3-Basic(Promega, Madison, Wis.) vector, which carries the firefly luciferasegene.

A DNA sequence corresponding to the TATA box of the miR-21 promoter (SEQID NO: 6) was constructed by annealing the following oligonucleotides:fw 5′-CCTAGTGGTGATAAATGTGGGACTTCTGAGAAGTCATTCATTTTATTCTTTGTGCCATACCAGAGTACAA-3′ (SEQ ID NO: 16) and 5′-TCGAAACATGAGACCATACCGTGTTTCTTATTTTACTTACTGAAGAGTCTTCAGGGTGTAAATAGTGGTGATCCCAT G-3′ (SEQ IDNO: 17). The resultant fragment was directly cloned into pGL3-Basic toyield the vector TATA-Luc (SEQ ID NO: 20)

To examine the selective expression of heterologous gene products undermiR-21 promoter control in cancerous cells, miR-21-Luc and its controlTATA-Luc were transfected into different transformed and untransformedcells and the luciferase expression levels of the transfected cell lineswere compared.

FIG. 2 demonstrates miR-21 promoter-driven expression of the luciferasegene in Chinese hamster ovary cells (CHO) and the human colorectalcancer cell lines HCT116 and COLO320. As can be seen from FIG. 2, thehuman cancer cell lines transfected with miR-21-Luc exhibitedsignificantly higher expression of luciferase, as compared to the cancercells transfected with the control vector TATA-Luc. In addition, the CHOcells transfected with either vector showed low levels of expression ofluciferase, similar to the levels exhibited by the cancer cellstransfected with the control vector TATA-Luc. These results indicatethat heterologous gene products may be expressed at high levels inmiR-21-expressing cells, such as certain cancers, using constructsencoding the heterologous gene under the control of an mir-21 promotersequence.

Example 3 miR-21 Promoter Driven “Killer Gene” Expression

To determine the ability of regulatory promoter sequences of miR-21 tospecifically drive the expression of a “killer gene”, the luciferasecoding regions of the reporter and control vectors described in Example2 were replaced with a nucleic acid sequence encoding the cytotoxicfragment A of diphtheria toxin (DT-A). DT-A inhibits protein synthesis,triggers apoptosis and detachment of cells, and has been previously usedas a “killer gene” for targeting cancer cells.

The DNA sequence encoding DT-A, and the corresponding amino acidsequence are provided herein as SEQ ID NO: 18 and SEQ ID NO: 19,respectively. The constructed plasmids containing the miR-21 promotersequence of SEQ ID NO:1 or its TATA box of SEQ ID NO: 6 wererespectively designated miR-21 Pr-DT-A (SEQ ID NO: 9) and miR-21 TATAbox DT-A (SEQ ID NO: 13).

Apoptosis levels and protein synthesis rates were determined to confirmthe miR-21 promoter-driven expression of DT-A. To determine thereduction in de novo protein synthesis, miR-21 Pr DT-A and a expressionvector containing Renilla luciferase under the control of an SV-40promoter were cotransfected into cells. As demonstrated in FIG. 3,transfectants of three different cancer cell types (Huh7, humanhepatocellular carcinoma; MCF7, human breast cancer; HCT116, human coloncarcinoma) containing miR-21 Pr DT-A showed strong reduction inluciferase activity, compared to the corresponding transfectantscontaining miR-21 TATA box DT-A, indicating the efficiency of the miR-21promoter-driven approach.

The next set of experiments was performed using a plasmid in which thenucleic acid sequence encoding the p53 up-regulated modulator ofapoptosis (PUMA) replaced the Luc sequence in the plasmids described inExample 2. Thus, the plasmid miR-21 pr PUMA (SEQ ID NO: 10), having thePUMA sequence under the control of the miR-21 promoter sequence of SEQID NO:1 was produced. miR-21 pr PUMA and miR-21 Pr DT-A wereco-transfected into cells together with a luciferase expressing plasmid.Forty eight hours post transfection the cells were harvested andluciferase activity was determined Reduction in de novo proteinsynthesis by DT-A and induction of apoptosis by PUMA were observed incells transfected with the miR-21 promoter driven plasmids, but not incells transfected with the TATA box control plasmids (FIG. 4A).

A similar experiment was carried out in which a plasmid encoding greenfluorescent protein (GFP; SEQ ID NO: 25 was co-transfected together withthe miR-21 promoter driven plasmids (miR-21 Pr DT-A or miR-21 pr PUMA)or the TATA box control plasmids (miR-21 TATA box DT-A or miR-21 TATAbox PUMA). After 48 hours the cells were lysed and the lysates subjectedto Western blot analysis with an anti-GFP antibody (FIG. 4B). As seen inFIG. 4B, the GFP signal was completely eliminated in the cotransfectionsystems with miR-21 DT-A and with miR-21 PUMA, but not in the systemswith the TATA box driven controls.

Example 4 miR-21 Promoter Driven DT-A Expression Inhibits TumorFormation in Vivo

Nude mice (n=3 in each group) were injected with 1×107 HCT116 cells. Twoweeks post injection tumor starting size was determined Two weeksfollowing tumor cell injection, endotoxin-free miR-21 Pr DT-A (25 ug)complexed with the transfection reagent JETPEI™ (Polyplus, France) wasinjected intratumorly. An additional injection was administered 7 dayslater. Tumor size was recorded at days 0, 7 and 14 using a manualcaliper. Injection of miR-21-Luc (described in Example 2) served as acontrol. In one mouse injected with miR-21 Pr DT-A, the tumor completelydisappeared. In another mouse similarly treated with miR-21 Pr DT-A, thetumor growth was inhibited but its size was not reduced. Overall, themean fold change for the treated mice was 1 compared to 1.6 in thecontrol mice, as shown in FIG. 5.

In additional experiments, the expression level of miR-21 may beexamined in cancer cells, such as cell lines of human hepatocellularcarcinoma, colorectal cancer, and breast cancer. Particularly preferredare cancer cells which have shown to be susceptible to the cytotoxiceffects of the constructs and vectors of the invention upon transfectionand expression of the anti-cancer agent encoded therein. Determinationof expression levels of miR-21 may be carried out using for example,real-time RT-PCR.

References

1. Ohana, P., P. Schachter, B. Ayesh, A. Mizrahi, T. Birman, T.Schneider, I. Matouk, S. Ayesh, P. J. Kuppen, N. de Groot, A. Czerniak,and A. Hochberg, Regulatory sequences of H19 and IGF2 genes in DNA-basedtherapy of colorectal rat liver metastases. J Gene Med, 2005. 7(3): p.366-374.

2. Varda-Bloom, N., I. Hodish, A. Shaish, S. Greenberger, R. Tal, B.Feder, J. Roitelman, E. Breitbart, L. Bangio, I. Barshack, R. Pfeffer,and D. Harats, Specific Induction of Tumor Neovasculature Death byModified Murine PPE-1 Promoter Armed with HSV-TK. Pathobiology, 2008.75(6): p. 346-355.

3. Giladi, N., H. Dvory-Sobol, E. Sagiv, D. Kazanov, E. Liberman, and N.Arber, Gene therapy approach in prostate cancer cells using an activeWnt signal. Biomedicine & Pharmacotherapy, 2007. 61(9): p. 527-530.

4. Barnes, D., M. Kunitomi, M. Vignuzzi, K. Saksela, and R. Andino,Harnessing Endogenous miRNAs to Control Virus Tissue Tropism as aStrategy for Developing Attenuated Virus Vaccines. Cell Host & Microbe,2008. 4(3): p. 239-248.

5. Chan, J. A., A. M. Krichevsky, and K. S. Kosik, MicroRNA-21 is anantiapoptotic factor in human glioblastoma cells. Cancer Res, 2005.65(14): p. 6029-6033.

6. Gabriely, G., T. Wurdinger, S. Kesari, C. C. Esau, J. Burchard, P. S.Linsley, and A. M. Krichevsky, MicroRNA 21 promotes glioma invasion bytargeting matrix metalloproteinase regulators. Mol Cell Biol, 2008.28(17): p. 5369-5380.

7. Iorio, M. V., M. Ferracin, C. G. Liu, A. Veronese, R. Spizzo, S.Sabbioni, E. Magri, M. Pedriali, M. Fabbri, M. Campiglio, S. Menard, J.P. Palazzo, A. Rosenberg, P. Musiani, S. Volinia, I. Nenci, G. A. Calin,P. Querzoli, M. Negrini, and C. M. Croce, MicroRNA gene expressionderegulation in human breast cancer. Cancer Res, 2005. 65(16): p.7065-7070.

8. Sempere, L. F., M. Christensen, A. Silahtaroglu, M. Bak, C. V. Heath,G. Schwartz, W. Wells, S. Kauppinen, and C. N. Cole, Altered MicroRNAexpression confined to specific epithelial cell subpopulations in breastcancer. Cancer Res, 2007. 67(24): p. 11612-11620.

9. Si, M. L., S. Zhu, H. Wu, Z. Lu, F. Wu, and Y. Y. Mo, miR-21-mediatedtumor growth. Oncogene, 2007. 26(19): p. 2799-2803.

10. Qian, B., D. Katsaros, L. Lu, M. Preti, A. Durando, R. Arisio, L.Mu, and H. Yu, High miR-21 expression in breast cancer associated withpoor disease-free survival in early stage disease and high TGF-beta1.Breast Cancer Res Treat, 2008.

11. Yan, L. X., X. F. Huang, Q. Shao, M. Y. Huang, L. Deng, Q. L. Wu, Y.X. Zeng, and J. Y. Shao, MicroRNA miR-21 overexpression in human breastcancer is associated with advanced clinical stage, lymph node metastasisand patient poor prognosis. RNA, 2008. 14(11): p. 2348-2360.

12. Kutay, H., S. Bai, J. Datta, T. Motiwala, I. Pogribny, W. Frankel,S. T. Jacob, and K. Ghoshal, Downregulation of miR-122 in the rodent andhuman hepatocellular carcinomas. J Cell Biochem, 2006. 99(3): p.671-678.

13. Connolly, E., M. Melegari, P. Landgraf, T. Tchaikovskaya, B. C.Tennant, B. L. Slagle, L. E. Rogler, M. Zavolan, T. Tuschl, and C. E.Rogler, Elevated expression of the miR-17-92 polycistron and miR-21 inhepadnavirus-associated hepatocellular carcinoma contributes to themalignant phenotype. Am J Pathol, 2008. 173(3): p. 856-864.

14. Jiang, J., Y. Gusev, I. Aderca, T. A. Mettler, D. M. Nagorney, D. J.Brackett, L. R. Roberts, and T. D. Schmittgen, Association of MicroRNAexpression in hepatocellular carcinomas with hepatitis infection,cirrhosis, and patient survival. Clin Cancer Res, 2008. 14(2): p.419-427.

15. Ladeiro, Y., G. Couchy, C. Balabaud, P. Bioulac-Sage, L. Pelletier,S. Rebouissou, and J. Zucman-Rossi, MicroRNA profiling in hepatocellulartumors is associated with clinical features and oncogene/tumorsuppressor gene mutations. Hepatology, 2008. 47(6): p. 1955-1963.

16. Meng, F., R. Henson, M. Lang, H. Wehbe, S. Maheshwari, J. T.Mendell, J. Jiang, T. D. Schmittgen, and T. Patel, Involvement of humanmicro-RNA in growth and response to chemotherapy in humancholangiocarcinoma cell lines. Gastroenterology, 2006. 130(7): p.2113-2129.

17. Roldo, C., E. Missiaglia, J. P. Hagan, M. Falconi, P. Capelli, S.Bersani, G. A. Calin, S. Volinia, C. G. Liu, A. Scarpa, and C. M. Croce,MicroRNA expression abnormalities in pancreatic endocrine and acinartumors are associated with distinctive pathologic features and clinicalbehavior. J Clin Oncol, 2006. 24(29): p. 4677-4684.

18. Lee, E. J., Y. Gusev, J. Jiang, G. J. Nuovo, M. R. Lerner, W. L.Frankel, D. L. Morgan, R. G. Postier, D. J. Brackett, and T. D.Schmittgen, Expression profiling identifies microRNA signature inpancreatic cancer. Int J Cancer, 2007. 120(5): p. 1046-1054.

19. Dillhoff, M., J. Liu, W. Frankel, C. Croce, and M. Bloomston,MicroRNA-21 is Overexpressed in Pancreatic Cancer and a PotentialPredictor of Survival. J Gastrointest Surg, 2008.

20. Volinia, S., G. A. Calin, C. G. Liu, S. Ambs, A. Cimmino, F.Petrocca, R. Visone, M. Iorio, C. Roldo, M. Ferracin, R. L. Prueitt, N.Yanaihara, G. Lanza, A. Scarpa, A. Vecchione, M. Negrini, C. C. Harris,and C. M. Croce, A microRNA expression signature of human solid tumorsdefines cancer gene targets. Proc Natl Acad Sci USA, 2006. 103(7): p.2257-2261.

21. Fulci, V., S. Chiaretti, M. Goldoni, G. Azzalin, N. Carucci, S.Tavolaro, L. Castellano, A. Magrelli, F. Citarella, M. Messina, R.Maggio, N. Peragine, S. Santangelo, F. R. Mauro, P. Landgraf, T. Tuschl,D. B. Weir, M. Chien, J. J. Russo, J. Ju, R. Sheridan, C. Sander, M.Zavolan, A. Guarini, R. Foa, and G. Macino, Quantitative technologiesestablish a novel microRNA profile of chronic lymphocytic leukemia.Blood, 2007. 109(11): p. 4944-4951.

22. Gao, J., T. T. Yang, X. C. Qiu, B. Yu, J. W. Han, Q. Y. Fan, and B.A. Ma, [Cloning and identification of microRNA from human osteosarcomacell line SOSP-9607]. Ai Zheng, 2007. 26(6): p. 561-565.

23. Iorio, M. V., R. Visone, G. Di Leva, V. Donati, F. Petrocca, P.Casalini, C. Taccioli, S. Volinia, C. G. Liu, H. Alder, G. A. Calin, S.Menard, and C. M. Croce, MicroRNA signatures in human ovarian cancer.Cancer Res, 2007. 67(18): p. 8699-8707.

24. Lawrie, C. H., S. Soneji, T. Marafioti, C. D. Cooper, S. Palazzo, J.C. Paterson, H. Cattan, T. Enver, R. Mager, J. Boultwood, J. S.Wainscoat, and C. S. Hatton, MicroRNA expression distinguishes betweengerminal center B cell-like and activated B cell-like subtypes ofdiffuse large B cell lymphoma. Int J Cancer, 2007. 121(5): p. 1156-1161.

25. Lawrie, C. H., S. Gal, H. M. Dunlop, B. Pushkaran, A. P. Liggins, K.Pulford, A. H. Banham, F. Pezzella, J. Boultwood, J. S. Wainscoat, C. S.Hatton, and A. L. Harris, Detection of elevated levels oftumour-associated microRNAs in serum of patients with diffuse largeB-cell lymphoma. Br J Haematol, 2008. 141(5): p. 672-675.

26. Lui, W. O., N. Pourmand, B. K. Patterson, and A. Fire, Patterns ofknown and novel small RNAs in human cervical cancer. Cancer Res, 2007.67(13): p. 6031-6043.

27. Loffler, D., K. Brocke-Heidrich, G. Pfeifer, C. Stocsits, J.Hackermuller, A. K. Kretzschmar, R. Burger, M. Gramatzki, C. Blumert, K.Bauer, H. Cvijic, A. K. Ullmann, P. F. Stadler, and F. Horn,Interleukin-6 dependent survival of multiple myeloma cells involves theStat3-mediated induction of microRNA-21 through a highly conservedenhancer. Blood, 2007. 110(4): p. 1330-1333.

28. Pichiorri, F., S. S. Suh, M. Ladetto, M. Kuehl, T. Palumbo, D.Drandi, C. Taccioli, N. Zanesi, H. Alder, J. P. Hagan, R. Munker, S.Volinia, M. Boccadoro, R. Garzon, A. Palumbo, R. I. Aqeilan, and C. M.Croce, MicroRNAs regulate critical genes associated with multiplemyeloma pathogenesis. Proc Natl Acad Sci USA, 2008. 105(35): p.12885-12890.

29. Slaby, O., M. Svoboda, P. Fabian, T. Smerdova, D. Knoflickova, M.Bednarikova, R. Nenutil, and R. Vyzula, Altered expression of miR-21,miR-31, miR-143 and miR-145 is related to clinicopathologic features ofcolorectal cancer. Oncology, 2007. 72(5-6): p. 397-402.

30. Chan, S. H., C. W. Wu, A. F. Li, C. W. Chi, and W. C. Lin, miR-21microRNA expression in human gastric carcinomas and its clinicalassociation. Anticancer Res, 2008. 28(2A): p. 907-911.

31. Schetter, A. J., S. Y. Leung, J. J. Sohn, K. A. Zanetti, E. D.Bowman, N. Yanaihara, S. T. Yuen, T. L. Chan, D. L. Kwong, G. K. Au, C.G. Liu, G. A. Calin, C. M. Croce, and C. C. Harris, MicroRNA expressionprofiles associated with prognosis and therapeutic outcome in colonadenocarcinoma. JAMA, 2008. 299(4): p. 425-436.

32. Zhang, Z., Z. Li, C. Gao, P. Chen, J. Chen, W. Liu, S. Xiao, and H.Lu, miR-21 plays a pivotal role in gastric cancer pathogenesis andprogression. Lab Invest, 2008.

33. Tran, N., T. McLean, X. Zhang, C. J. Zhao, J. M. Thomson, C.O'Brien, and B. Rose, MicroRNA expression profiles in head and neckcancer cell lines. Biochem Biophys Res Commun, 2007. 358(1): p. 12-17.

34. Chang, S. S., W. W. Jiang, I. Smith, L. M. Poeta, S. Begum, C.Glazer, S. Shan, W. Westra, D. Sidransky, and J. A. Califano, MicroRNAalterations in head and neck squamous cell carcinoma. Int J Cancer,2008. 123(12): p. 2791-2797.

35. Feber, A., L. Xi, J. D. Luketich, A. Pennathur, R. J. Landreneau, M.Wu, S. J. Swanson, T. E. Godfrey, and V. R. Litle, MicroRNA expressionprofiles of esophageal cancer. J Thorac Cardiovasc Surg, 2008. 135(2):p. 255-260; discussion 260.

36. Wang, T., X. Zhang, L. Obijuru, J. Laser, V. Aris, P. Lee, K.Mittal, P. Soteropoulos, and J. J. Wei, A micro-RNA signature associatedwith race, tumor size, and target gene activity in human uterineleiomyomas. Genes Chromosomes Cancer, 2007. 46(4): p. 336-347.

37. Amaral, F. C., N. Torres, F. Saggioro, L. Neder, H. R. Machado, W.A. Silva, Jr., A. C. Moreira, and M. Castro, MicroRNAs differentiallyexpressed in ACTH-secreting pituitary tumors. J Clin Endocrinol Metab,2008.

38. Markou, A., E. G. Tsaroucha, L. Kaklamanis, M. Fotinou, V.Georgoulias, and E. S. Lianidou, Prognostic value of mature microRNA-21and microRNA-205 overexpression in non-small cell lung cancer byquantitative real-time RT-PCR. Clin Chem, 2008. 54(10): p. 1696-1704.

39. Nam, E. J., H. Yoon, S. W. Kim, H. Kim, Y. T. Kim, J. H. Kim, J. W.Kim, and S. Kim, MicroRNA expression profiles in serous ovariancarcinoma. Clin Cancer Res, 2008. 14(9): p. 2690-2695.

40. Neely, L. A., K. M. Rieger-Christ, B. S, Neto, A. Eroshkin, J.Garver, S. Patel, N. A. Phung, S. McLaughlin, J. A. Libertino, D.Whitney, and I. C. Summerhayes, A microRNA expression ratio defining theinvasive phenotype in bladder tumors. Urol Oncol, 2008.

41. Betel, D., M. Wilson, A. Gabow, D. S. Marks, and C. Sander, ThemicroRNA.org resource: targets and expression. Nucl. Acids Res., 2008.36(suppl_(—)1): p. D149-153.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

The invention claimed is:
 1. A nucleic acid construct comprising anmiR-21 promoter sequence and at least one nucleic acid sequence encodinga cytotoxic agent, wherein the nucleic acid sequence encoding thecytotoxic agent is operably linked to the miR-21 promoter sequence,wherein the miR-21 promoter sequence has at least 80% identity with SEQID NO:
 1. 2. The construct of claim 1, wherein the miR-21 promotersequence comprises SEQ ID NO:
 1. 3. The construct of claim 1, whereinthe cytotoxic agent is selected from the group consisting of a toxin, afragment of a toxin, a drug-metabolizing enzyme, and an inducer ofapoptosis.
 4. The construct of claim 1, wherein said cytotoxic agent isa toxin selected from the group consisting of a bacterial toxin, a planttoxin, a fungal toxin and a combination thereof; a drug-metabolizingenzyme comprising a kinase; or an inducer of apoptosis selected from thegroup consisting of PUMA; BAX; BAK; Bci-XS; BAD; BIM; BIK; BID; HRK; AdEIB; an ICE-CED3 protease; TRAIL; SARP-2; and apoptin.
 5. The constructof claim 4, wherein said cytotoxic agent is a toxin consisting of abacterial toxin selected from the group consisting of diphtheria toxin,Pseudomonas exotoxin, cholera toxin, anthrax toxin, botulinum toxin,pertussis toxin, E. coli enterotoxin, and shiga toxin; a plant toxinselected from the group consisting of ricin, modeccin, abrin, volkensinand viscumin; or a fungal toxin selected from the group consisting of asarcin, restrictocin , mitogillin, enomycin, RNase T1 and phenomycin. 6.The construct of claim 5, wherein said toxin is a diphtheria toxinconsisting of fragment A of diphtheria toxin (DT-A) and having the aminoacid sequence of SEQ ID NO:
 19. 7. The construct of claim 1, comprisingSEQ ID NO: 1 as the miR-21 promoter sequence, and further comprising SEQID NO: 18 as the nucleic acid sequence encoding a cytotoxic agent. 8.The construct of claim 1, wherein the nucleic acid construct issubstantially devoid of a nucleic acid sequence corresponding to orcomplementary to a form of miR-21 selected from the group consisting of:a primary transcript ofmiR-21 (pri-miR-21); a precursor of miR-21(pre-miR-21); an RNA duplex of miR-21, and a mature miR-21.
 9. Theconstruct of claim 1, comprising a sequence selected from the groupconsisting of SEQ ID NO: 7 and SEQ ID NO:
 8. 10. A vector comprising thenucleic acid construct of claim
 1. 11. The vector of claim 10, selectedfrom the group consisting of SEQ ID NO: 9 and SEQ ID NO:
 10. 12. Anisolated host cell comprising the vector of claim
 10. 13. Apharmaceutical composition comprising as an active ingredient theconstruct of claim 1, and at least one pharmaceutically acceptablecarrier, excipient or diluent.
 14. A kit comprising; i) one or moredosage units of the vector of claim 10; and ii) instructions foradministering said nucleic acid construct to a subject in need thereof.